Partially loaded antibodies and methods of their conjugation

ABSTRACT

A protein containing one or more activatable groups, e.g., an antibody, is subjected to partial or complete reduction of one or more such bonds to form reactive groups; the resulting protein is reacted with a drug which is reactive with some of the reactive groups, such as certain radiometals, chelating agents, and toxins, so as to form a conjugate useful in, e.g., in vitro diagnosis, in vivo imaging, and therapy.

CONTINUITY

This application claims the benefit of U.S. Provisional Application No.60/549,476, filed Mar. 2, 2004, the disclosure of which is incorporatedby reference herein in its entirety.

BACKGROUND

The present invention is directed to modified proteins having at leastone point of conjugation with, for example, a drug resulting in specificisomers of the protein-drug conjugate, and to methods for suchconjugation resulting in the specific isomers. The invention is furtherdirected to antibodies to which cytotoxic agents and/or cytostaticagents can be conjugated resulting in specific isomers and methods fortheir conjugation.

Monoclonal antibodies (mAbs) are a valuable weapon in the battle againstcancer. mAbs are also used in the treatment of immune disorders. Tofurther advance the use of mAb-based therapies for cancer and immunedisorders, a number of novel approaches have been explored. One approachis to increase the cytotoxic potential of mAbs against tumor cells byattaching cell-killing payloads. Molecules such as protein toxins,radionuclides, and anti-cancer drugs have been conjugated to certainmAbs to generate immunotoxins, radioimmunoconjugates, and antibody-drugconjugates (ADCs), respectively.

Factors which have previously been considered in developing ADCs haveincluded the choice of antibody, and optimizing the potency of the drugcomponent, the stability of the linker, and the method by which the drugwas covalently attached to the mAb. The common convention for producingADCs conjugated through the disulfide bonds has been by reducing allinter-chain disulfide bonds of an antibody and reacting all the reducedmAb thiols with a compound capable of interaction with all the reducedthiols, forming uniformly-substituted ADCs with 8 drugs/mAb, i.e. “fullyloaded,” without the ability to obtain specificity for certain site ofconjugation.

For example, the antigen CD30 is highly expressed on cancers such asHodgkin's disease (HD) and anaplastic large cell lymphomas (ALCL). Thisexpression of CD30, coupled with limited expression on normal cells,makes it an attractive target for ADC therapy. The chimeric mAb directedto CD30, cAC10, has antitumor activity against HD both in vitro and insubcutaneous and disseminated SCID mouse xenograft models. Theanti-tumor activity of cAC10 was enhanced by generating fully loadedADCs in which all eight of the interchain thiols were linked toderivatives of the cytotoxic agent auristatin E as the drug component.These ADCs were highly effective in murine xenograft models atwell-tolerated doses.

Because the convention in the production of ADCs has been to fully loadthem with drug, it was not previously appreciated that partially-loadedADCs could have the same or greater therapeutic efficacy. Further,methods did not exist which could take into consideration that othersubstitution patterns on antibodies could produce equal or bettertherapeutic efficacy with equal or lower toxicity. These and otherlimitations and problems of the past are solved by the presentinvention.

BRIEF SUMMARY

The present invention provides protein-drug conjugates and methods ofmaking protein-drug and protein-labeled conjugates. Also provided areproteins having points of conjugation for receiving a drug or label. Theconjugates can be used therapeutically, diagnostically (e.g., in vitroor in vivo), for in vivo imaging, and for other uses.

Generally, partially loaded, modified protein having assignableconjugation points are provided. The modified proteins generally includea binding region for interaction with a binding partner and at least twopoints of conjugation, each point of conjugation covalently linked adrug or label. Typically, less that all possible points of conjugationhaving a similar accessibility or activability are linked to a drug orlabel. The modified protein can be, for example, an antibody, areceptor, a receptor ligand, a hormone, a cytokine, or the like. Thepoints of conjugation can be, for example, amino groups, vicinalhydroxyl groups, hydroxyl groups, carboxyl groups, or thiol groups. Theprotein can be, for example, a receptor, a receptor ligand, a hormone, acytokine, or the like.

In further embodiments, a method of preparing a conjugate of a proteinhaving one or more disulfide bonds, and a drug reactive with free thiolsis provided. The method generally includes partially reducing theprotein with a reducing agent; and conjugating the drug reactive withfree thiols to the partially reduced protein. In yet another embodiment,a method of preparing a conjugate of a protein having one or moredisulfide bonds, and a drug reactive with free thiols, is provided. Themethod generally includes fully reducing the protein with a reducingagent; partially reoxidizing the protein with a reoxidizing agent; andconjugating the drug reactive with free thiols to the antibody.

In some embodiments, a partially-loaded antibody is provided. Theantibody includes an antigen binding region, at least one interchaindisulfide bond, and at least two drugs or labels, each drug or labelconjugated to an interchain thiol. The points of the conjugation of thedrug or label optionally are readily assignable. In an example, theantibody can have at least four cytotoxic or cytostatic drugs, each drugconjugated to an interchain thiol. In certain embodiments, the antibodyhas the configuration of species 4A, 4B, 4C, 4D, 4E or 4F.

The partially-loaded antibody can be, for example, a murine, humanized,human or chimeric antibody. The drug can be, for example, a cytotoxic orcytostatic agent such as, for example, MMAF, MMAE or AFP. Also providedare pharmaceutical compositions comprising partially loaded antibodies.

In anther embodiment, antibodies are provided having at least one pointof conjugation for a cytotoxic or cytostatic agent, wherein the point ofconjugation for the cytotoxic or cytostatic agent on the antibody can bereadily assigned. On the antibody, less than all possible points areconjugation are available for conjugation to the cytotoxic or cytostaticagent. The points of conjugation can be, for example, interchain thiols.The points of conjugation can be, for example, at least one of species4A through 4F.

A composition of modified antibodies having assignable conjugationpoints is also provided. The composition can have, for example, at leasttwo, at least four, at least 6 at least 7, at least 10 or more speciesof modified antibody. In one example, each species can have at least onespecified conjugation pairs having two interchain thiols, and at leastone interchain disulfide bond. The antibody species can have, forexample, 4A, 4B, 4C, 4D, 4E and/or 4F. In further examples, thespecified conjugation pair can be at a constant light-constant heavyinterchain disulfide bond and/or at a constant heavy-constant heavyinterchain disulfide bond. The specified conjugation pair can beproximal to the N-terminal end of the hinge region and/or proximal tothe C-terminal end of the hinge region. In another example, thespecified conjugation pair is at the constant light-constant heavyinterchain sulfide bond and at the hinge region located closer to theN-terminal end of the modified antibody, or at the constantlight-constant heavy interchain disulfide bond and at the hinge regionlocated closer to the C-terminal end of the modified antibody.

Each species of antibody optionally can include at least two specifiedconjugation pairs at the constant light-constant heavy interchaindisulfide bonds or at least two specified conjugation pairs at the hingeregion interchain disulfide bonds. The composition optionally canfurther include a pharmaceutically acceptable carrier.

In yet another embodiment, a partially loaded antibody is provided. Theantibody includes at least one antigen-binding domain, at least tworeactive group on the antibody, and at least two drugs or labels, eachdrug or label conjugated to a reactive group to form a point ofconjugation. The points of conjugation for the drug or label are readilyassignable.

In yet other embodiments, a method of reducing and conjugating a drug toan antibody resulting in selectivity in the placement of the drug isprovided. The method generally includes fully reducing the antibody witha reducing agent, treating the fully reduced antibody with limitingamounts of a reoxidizing agent to reform at least one interchaindisulfide bond of the antibody, such that at least two interchain thiolsremain; and conjugating the drug to the interchain thiols. Thereoxidizing agent can be, for example, 5,5′-dithio-bis-2-nitrobenzoicacid, 4,4′-dithiodipyridine, 2,2′-dithiodipyridine, sodium tetrathionateor iodosobenzoic acid. The drug can be, for example, a cytotoxic orcytostatic agent or an immunosuppressive agent. In some examples, thedrug can be a minor grove binder, AEB, AEVB, MMAF, MMAE or AFP. Thereducing agent can be, for example, DTT or TCEP.

In related embodiments, a method of reducing antibody interchaindisulfide bonds and conjugating a drug to the resulting interchainthiols resulting in selectivity in the placement of the drugs on theantibody is provided. The method generally includes fully reducing theantibody with a reducing agent to form interchain thiols; partiallyreoxidizing the antibody with a reoxidizing agent to reform at least oneinterchain disulfide bond; and conjugating the drug to the interchainthiols. The reoxidizing agent can be, for example,5,5′-dithio-bis-2-nitrobenzoic acid, 4,4′-dithiodipyridine,2,2′-dithiodipyridine, sodium tetrathionate, or iodosobenzoic acid. Thereducing agent can be, for example, DTT or TCEP. The drug can be, forexample, MMAF, MMAE, or AFP. The partially reoxidized antibodyoptionally can be purified prior to conjugation.

In yet other related embodiments, a method of reducing antibodyinterchain disulfide bonds and conjugating a drug to the resultinginterchain thiols to selectively locate drugs on the antibody isprovided. The method generally includes partially reducing the antibodywith a reducing agent to form at least two interchain thiols; andconjugating the drug to the interchain thiols of the partially reducedantibody. In an example, the antibody is partially reduced with alimiting concentration of a reducing agent in a buffer with a chelatingagent. The drug can be conjugated, for example, by cooling the antibodysolution and dissolving the drug in a cold solvent and mixing with theantibody solution. The antibody and drug solution are incubated for aperiod of time sufficient to form a partially loaded antibody-drugconjugate(s). The reaction can be quenched with a quenching the excessdrug with a thiol-containing reagent. The conjugate can be furtherpurified. In a specific example, the antibody is partially reduced forabout 1 hour at about 37° C. The reduced antibody can be cooled, forexample, to about 0° C. The antibody and drug solution can be incubated,for example, for about 30 minutes at about 0° C.

The thiol-containing reagent can be, for example, cysteine or N-acetylcysteine. The reducing agent can be, for example, DTT or TCEP. Thebuffer can be, for example, a sodium borate solution and the chelatingagent is dethylenetriaminepentaacetic acid. The chelating agent also canbe, for example, ethylenetriaminepentaacetic acid or EDTA. The solventcan be, for example, acetonitrile, alcohol or DMSO. The drug can be, forexample, a cytotoxic or a cytostatic agent.

In some embodiments, the reduced antibody can be purified prior toconjugation, using for example, column chromatography, dialysis, ordiafiltration. The column used in column chromatography can be, forexample, a desalting column, such as a PD-10 column. Alternatively, thereduced antibody is not purified after partial reduction and prior toconjugation.

The conjugate can be purified using, for example, column chromatography,dialysis, or diafiltration. The column used in column chromatography canbe, for example, a desalting column, such as a PD-10 column.

In yet another embodiment, a method of producing an antibody withselective conjugation of drug is provided. The method generally includesfully reducing the antibody for a period of time sufficient to produceinterchain thiols, as determined by, for example, DTNB titration, byadding a large excess of a reducing agent and incubating the solution atabout 37° C. for about 30 minutes; purifying the antibody; partiallyreoxidizing the antibody using an oxidizing agent to form at least oneinterchain disulfide bond by cooling the reduced antibody to 0° C.;treating the reduced and cooled antibody with 1.5 to 2.5 molarequivalents of the oxidizing agent; mixing the solution by inversion;allowing the solution to incubate at about 0° C. for about 10 minutes;purifying the partially reoxidized antibody; conjugating the drug to theinterchain thiols of the partially reoxidized antibody to form aconjugated antibody; and purifying the conjugated antibody.

The reducing agent can be, for example, DTT or TCEP. Thepartially-reduced antibody optionally can be purified, for example,using column chromatography, dialysis, or diafiltration. The column usedin column chromatography can be, for example, a desalting column such asas a PD-10 column. The conjugated antibody can be purified, for example,using column chromatography, dialysis, or diafiltration. The column usedin column chromatography can be, for example, a desalting column such asas a PD-10 column. The reoxidizing agent can be, for example,5,5′-dithio-bis-2-nitrobenzoic acid, 4,4′-dithiodipyridine,2,2′-dithiodipyridine, sodium tetrathionate, or iodosobenzoic acid.

The invention will best be understood by reference to the followingdetailed description of the specific embodiments, taken in conjunctionwith the accompanying drawings. The discussion below is descriptive,illustrative and exemplary and is not to be taken as limiting the scopedefined by any appended claims.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows the “E4” isomers (isomers with four drugs attached perantibody) of a cartoon antibody. The interchain disulfide bonds areshown as solid lines between the heavy-heavy chains of the antibody orthe heavy-light chains of the antibody. The drugs and their points ofconjugation to the antibody are shown as circles. The fragmentsgenerated under non-reducing (“Non-red”) and reducing (“Red”) conditionsare shown below each isomer (with the number of drugs per fragment inparentheses).

FIG. 2 shows a chromatogram of the elution profile of a hydrophobicinteraction chromatography (HIC) analysis of vcMMAE-cAC10 conjugateprepared by one aspect of method 2a: partial DTNB reoxidation, PD-10purification, and vcMMAE conjugation. “E0”, “E2”, “E4”, “E6” and “E8”refer to the isomers of the cAC10 antibody with 0, 2, 4, 6 and 8 MMAFmolecules attached per antibody, respectively.

FIG. 3 shows a HIC chromatogram for another aspect of method 2b: one-potDTNB reoxidation and vcMMAE conjugation. E0”, “E2”, “E4”, “E6” and “E8”refer to the isomers of the cAC10 antibody with 0, 2, 4, 6 and 8 MMAFmolecules attached per antibody, respectively. Pure E4 was collectedfrom about 34-38 min (indicated by arrow).

FIG. 4 shows bar graph comparison of the percent composition ofantibody-drug conjugates for even drug loaded species from methods 1,2a, and 2b. For each species, antibody-drug conjugates were prepared bythe DTT partial reduction (left bar), DTNB reoxidation (middle bar) orone pot DTNB reoxidation (right bar) conjugation methods.

FIG. 5 shows a bioanalyzer trace for E4 material collected as indicatedin the HIC chromatogram in FIG. 3 (method 2b). “L” indicates free lightchains. “H” indicates free heavy chains. “HL” indicates associatedheavy-light chains. “HH” indicates associated heavy-heavy chains. “HHL”indicates associated heavy-heavy and heavy-light chains.

FIG. 6 shows PLRP analysis of material collected from the HICchromatogram in FIG. 3 (method 2b). “L0” and “L1” indicate a light chainwith no or one drug molecule attached, respectively. “H0”, “H1”, “H2”and “H3” indicate a heavy chain with zero, one, two or three drugmolecules attached.

FIG. 7 shows a cartoon representation of the major conjugate speciesobtained by reduction, full or partial, of the inter-chain disulfidebonds followed by conjugation with a drug. Full reduction andconjugation produces primarily the fully-loaded species with 8 drugs perantibody, whereas partial reduction and conjugation can lead to thegeneration of all the shown species. There is only one isomer each forthe 0 and 8 drug-loaded species, whereas the 2, 4 and 6 drug-loadedspecies contain 3, 4 and 3 isomers, respectively. (Referring to FIG. 1,note that species 4A is a mirror image of species 4C and species 4B is amirror image of species 4D. In FIG. 7, species 4A and 4C are referred toas 4A, and species 4B and 4D are referred to as species 4B.) Theinterchain disulfide bonds are shown as solid lines between theheavy-heavy chains or the heavy-light chains of the antibody. The drugsand their point of conjugation to the antibody are shown as circles.

FIG. 8 is a process flow diagram for one aspect of a “Partial Reduction”conjugation process using DTT to produce E4 mixed isomers (E4M). The pHof the cAC10 antibody is adjusted to 7.5 with sodium phosphate dibasicand EDTA is added to a final concentration of 5 mM. The antibodysolution is then heated to 37° C. To partially reduce the antibody, 2.95molar equivalents of DTT is added to the antibody solution and allowedto reduce for 105 min at 37° C. After reduction, the antibody solutionis cooled down to 2-8° C. and the excess DTT removed by constant-volumeultrafiltration/-diafiltraton (UF/DF) to obtain the reduced, purifiedcAC10. A sample of the reduced, purified cAC10 is taken and the thiolconcentration, the antibody concentration, and the thiol-to-antibodymolar ratio determined by A280 and DTNB tests. A slight excess of thedrug-linker vcMMAE (typically 2-15% excess in the form of a DMSOsolution) is then added into the antibody solution to start theconjugation reaction. The conjugation reaction is allowed to proceed for30 min at 2-8° C. to obtain the crude E4M. At the end of the conjugationreaction, any excess vcMMAE drug-linker is quenched by reacting with alarge excess of cysteine for 15 min at 2-8° C. to obtain the quenched,crude E4M. Buffer-exchange and removal of free drug and othersmall-molecule species is performed by constant-volume UF/DF (typically6-10 diavolumes) to obtain the E4M drug substance.

FIG. 9 is a process flow diagram for another aspect of a “PartialReduction” conjugation process using TCEP, in which an intermediatepurification step is not used, to produce E4 mixed isomers (E4M). The pHof the cAC10 antibody is adjusted to 7.5 with sodium phosphate dibasicand EDTA is added to a final concentration of 5 mM. The antibodysolution is then heated to 37° C. To partially reduce the antibody, 2.20molar equivalents of TCEP is added to the antibody solution and allowedto reduce for 105 min at 37° C. A sample of the reduction reaction istaken and the thiol concentration, the antibody concentration, and thethiol-to-antibody molar ratio determined by A280 and DTNB tests. Afterreduction, the antibody solution is cooled down to 2-8° C. A slightexcess of the drug-linker vcMMAE (typically 2-15% excess in the form ofa DMSO solution) is then added into the antibody solution to start theconjugation reaction. The conjugation reaction is allowed to proceed for20 min at 2-8° C. to obtain the crude E4M. At the end of the conjugationreaction, any excess vcMMAE drug-linker is quenched by reacting with alarge excess of N-acetyl cysteine for 20 min at 2-8° C. to obtain thequenched, crude E4M. Buffer-exchange and removal of free drug and othersmall-molecule species is performed by constant-volume UF/DF (typically6-10 diavolumes) to obtain the E4M drug substance.

FIG. 10 shows a graph of the internalization of cAC10-conjugatedantibody by CD30⁺ Karpas-299 cells. The cells were combined with 1 μg/mLof fluorescently-labeled cAC10 and serial dilutions of either cAC10,cAC10-E2, cAC10-E4, or cAC10-E8 from 20 μg/mL to 9 ng/mL. Afterincubation of the cells with the antibody, the labeled cells were washedwith staining media, and the fluorescence was measured. The normalizedfluorescence intensities were plotted versus mAb concentration asdescribed in Example 8.

FIGS. 11A and 11B show graphs of the internalization of cAC10 andcAC10-conjugated antibodies by CD30⁺ cells: A) Karpas-299 and B) L540cycells were incubated with serial dilutions of cAC10 and E2, E4 and E8species of cAC10 ADCs. Following a 96-hour incubation with the samples,[³H]-TdR was added and its incorporation was measured. The radioactivityof the treated samples was normalized to the untreated controls andplotted versus concentration.

FIGS. 12A and 12B show in vivo efficacy cAC10 and cAC10-conjugatedantibodies in SCID mice bearing subcutaneous xenografts. FIG. 12A showsthe results with SCID mice bearing Karpas-299 subcutaneous tumorsinjected with cAC10-E2 at 0.5 mg/kg or 1.0 mg/kg every four days forfour injections. cAC10-E4 and cAC10-8 were dosed at either 0.25 or 0.5mg/kg every four days for four injections. FIG. 12B shows the resultswith SCID mice with Karpas-299 subcutaneous tumors were treated with asingle dose of E2, E4 or E8 at 1.0 mg/kg.

FIGS. 13A-D show hydrophobic interaction chromatography HPLC traces of(A) E4 mix; (B) E2 pure made by preparative HIC; (C) E4 pure made bypreparative HIC; and (D) E6 pure made by preparative HIC, respectively.Samples were made by DTT partial reduction followed by MMAE conjugation.Chromatograms were normalized to the height of the tallest peak in eachchromatogram. Injections were 50 μL of 5-10 mg/mL cAC10-vcMMAE in PBSmixed with 50 μL of 2.0 M NaCl and 50 mM sodium phosphate pH 7.Separations were performed at 30° C.

FIG. 14 show PLRP-S HPLC traces of (A) E4 mix made by partial DTTreduction followed by MMAE conjugation (top trace); (B) E4 mix made byDTNB partial reoxidation followed by MMAE conjugation (second trace fromtop); (C) E4 pure made by partial DTT reduction followed by MMAEconjugation and purified by preparative HIC (second trace from bottom);and (D) E4 pure made by partial DTNB reoxidation followed by MMAEconjugation and purified by preparative HIC (bottom trace). Injectionswere 20 μL of 1 mg/mL cAC10-vcMMAE treated with 20 mM DTT for 15 min at37° C. Separations were performed at 80° C.

FIG. 15 shows Bioanalyzer (capillary electrophoresis) traces of (A) E4mix made by partial DTT reduction followed by MMAE conjugation (toptrace); (B) E4 mix made by DTNB partial reoxidation followed by MMAEconjugation (second trace from top); (C) E4 pure made by partial DTTreduction followed by MMAE conjugation and purified by preparative HIC(second trace from bottom); and (D) E4 pure made by partial DTNBreoxidation followed by MMAE conjugation and purified by preparative HIC(bottom trace). Samples were prepared under non-reducing conditions asdirected by the manufacturer.

DETAILED DESCRIPTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections whichfollow.

I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art pertinent to the methods and compositions described. As usedherein, the following terms and phrases have the meanings ascribed tothem unless specified otherwise.

The term “drug” as used herein means an element, compound, agent, ormolecular entity, including, e.g., a pharmaceutical, therapeutic, orpharmacologic compound. Drugs can be natural or synthetic or acombination thereof. A “therapeutic drug” is an agent that exerts atherapeutic (e.g., beneficial) effect on cancer cells or immune cells(e.g., activated immune cells), either alone or in combination withanother agent (e.g., a prodrug converting enzyme in combination with aprodrug). Typically, therapeutic drugs useful in accordance with themethods and compositions described herein are those that exert acytotoxic, cytostatic, or immunosuppressive effect. In certainembodiments, a drug is not a radioactive element.

“Cytotoxic agent,” in reference to the effect of an agent on a cell,means killing of the cell.

“Cytostatic agent” means an inhibition of cell proliferation.

The term “polypeptide” refers to a polymer of amino acids and itsequivalent and does not refer to a specific length of a product; thus,“peptides” and “proteins” are included within the definition of apolypeptide. Also included within the definition of polypeptides are“antibodies” as defined herein. A “polypeptide region” refers to asegment of a polypeptide, which segment may contain, for example, one ormore domains or motifs (e.g., a polypeptide region of an antibody cancontain, for example, one or more CDRs). The term “fragment” refers to aportion of a polypeptide typically having at least 20 contiguous or atleast 50 contiguous amino acids of the polypeptide. A “derivative”includes a polypeptide or fragment thereof having conservative aminoacid substitutions relative to a second polypeptide; or a polypeptide orfragment thereof that is modified by covalent attachment of a secondmolecule such as, e.g., by attachment of a heterologous polypeptide, orby glycosylation, acetylation, phosphorylation, and the like. Furtherincluded are, for example, polypeptide analogs containing one or moreanalogs of an amino acid (e.g., unnatural amino acids and the like),polypeptides with unsubstituted linkages, as well as other modificationsknown in the art, both naturally and non-naturally occurring.Polypeptide analogs include, for example, protein mimetics and bombesin.

The term “antibody” as used herein refers to (a) immunoglobulinpolypeptides and immunologically active portions of immunoglobulinpolypeptides, i.e., polypeptides of the immunoglobulin family, orfragments thereof, that contain an antigen binding site thatimmunospecifically binds to a specific antigen, or (b) conservativelysubstituted derivatives of such immunoglobulin polypeptides or fragmentsthat immunospecifically bind to the antigen. Antibodies are generallydescribed in, for example, Harlow & Lane, Antibodies: A LaboratoryManual (Cold Spring Harbor Laboratory Press, 1988).

An “antibody derivative” as used herein means an antibody, as definedabove, that is modified by covalent attachment of a heterologousmolecule such as, e.g., by attachment of a heterologous polypeptide, orby glycosylation, acetylation or phosphorylation not normally associatedwith the antibody, and the like.

The term “monoclonal antibody” refers to an antibody that is derivedfrom a single cell clone, including any eukaryotic or prokaryotic cellclone, or a phage clone, and not the method by which it is produced.Thus, the term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology.

The term “interchain disulfide bond,” in the context of an antibody,refers to a disulfide bond between two heavy chains, or a heavy and alight chain.

The term “interchain thiol” refers to a thiol group of an antibody heavyor light chain that can participate in the formation of an interchaindisulfide bond.

A protein is referred to as “fully-loaded” when all points ofconjugation of a particular type and/or of similar reactivity areconjugated to drugs, resulting in a homogeneous population ofprotein-drug conjugate. A protein is referred to as “partially-loaded”when only some of the possible points of conjugation of a particulartype and/or of a similar reactivity are conjugated to drugs, resultingin formation of a certain isomer or isomers of the protein-drugconjugate.

The term “isolated,” in the context of a molecule or macromolecule(e.g., an antibody) is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with the desired use (e.g., diagnostic or therapeutic)of the molecule, and may include enzymes, hormones, and otherproteinaceous or nonproteinaceous solutes. In some embodiments, anisolated molecule or macromolecule will be purified (1) to greater than95%, or greater than 99%, by weight of the molecule or macromolecule asdetermined by, for example, the Lowry or Bradford methods, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated moleculesand macromolecules include the molecule and macromolecule in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, isolated antibodywill be prepared by at least one purification step.

The abbreviation “AFP” refers todimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylenediaminehaving the general formula shown immediately following:

The abbreviation “MMAE” refers to monomethyl auristatin E having thegeneral formula shown immediately following:

The abbreviation “MMAF” refers todovaline-valine-dolaisoleunine-dolaproine-phenylalanine having thegeneral formula shown immediately following:

The abbreviation “AEB” refers to an ester produced by reactingauristatin E with paraacetyl benzoic acid. The abbreviation “AEVB”refers to an ester produced by reacting auristatin E with benzoylvalericacid.

The phrase “pharmaceutically acceptable salt,” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a molecule ormacromolecule. Acid addition salts can be formed with amino groups.Exemplary salts include, but are not limited, to sulfate, citrate,acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate,phosphate, acid phosphate, isonicotinate, lactate, salicylate, acidcitrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucuronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate(i.e., 1,1′ methylene bis-(2-hydroxy 3-naphthoate))salts. Apharmaceutically acceptable salt may involve the inclusion of anothermolecule such as an acetate ion, a succinate ion or other counterion.The counterion may be any organic or inorganic moiety that stabilizesthe charge on the parent compound. Furthermore, a pharmaceuticallyacceptable salt may have more than one charged atom in its structure.Instances where multiple charged atoms are part of the pharmaceuticallyacceptable salt can have multiple counter ions. Hence, apharmaceutically acceptable salt can have one or more charged atomsand/or one or more counterion.

“Pharmaceutically acceptable solvate” or “solvate” refer to anassociation of one or more solvent molecules and a molecule ormacromolecule. Examples of solvents that form pharmaceuticallyacceptable solvates include, but are not limited to, water, isopropanol,ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

II. Polypeptides, Proteins, Antibodies

The present invention provides protein-drug conjugates and methods ofmaking protein-drug conjugates. Also provided are proteins having pointsof conjugation for receiving a drug. The protein-drug conjugates can beused therapeutically, diagnostically (e.g., in vitro or in vivo), for invivo imaging, and for other uses.

Various classes of proteins can be conjugated, including antibodies,enzymes, glycosylated proteins, lectins, various biological receptors,protein hormones, and other proteins that can serve as a binding agentfor a binding partner. The proteins contain at least one reactive site,such as a disulfide bond, amino group, hydroxyl group or carboxyl group,where conjugation of a drug to the protein can occur.

The reactive site is accessible and capable of activation, such as bychemical or means. In some embodiments, the protein to be chemicallyactivated for conjugation purposes is one containing disulfide bondsnon-essential for the intended use of the protein, and/or one whichwould not interfere with the protein (such as but not limited to causingdegradation of the protein or interfere with binding or other functions(e.g., effector function.)). In such a protein, a disulfide bond ispresent as a result of the oxidation of the thiol (—SH) side groups oftwo cysteine residues. These residues may lie on different polypeptidechains, or on the same polypeptide chain. As a result of the oxidation,a disulfide bond (—S—S—) is formed between the beta carbons of theoriginal cysteine residues. After reduction, the residues are termedoften interchangeably half-cystines and cystine. Treatment of thedisulfide bond with a reducing agent causes reductive cleavage of thedisulfide bonds to leave free thiol groups. Examples of proteinscontaining disulfide bonds include antibodies, many enzymes, certainhormones, and certain receptors.

In some embodiments, the disulfide bond can be naturally occurring. Insome embodiments, a sulfhydryl group(s) can also be chemicallyintroduced into a protein (e.g., an antibody). Suitable methods forintroducing sulfhydryl groups include chemical means (e.g., using athiolating agent such as 2-IT), or using recombinant DNA technology. Forexample, cysteine residues can be introduced into a protein bymutagenesis of a nucleic acid encoding the protein. See generallySambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed., ColdSpring Harbor Publish., Cold Spring Harbor, N.Y. (2001); Ausubel et al.,Current Protocols in Molecular Biology, 4th ed., John Wiley and Sons,New York (1999); which are incorporated by reference herein.) Sulfhydrylgroups can be introduced into a protein, for example, within thepolypeptide or at the carboxy-terminus.

In some embodiments, the protein is an antibody. Such an antibody may beused in in vitro or in vivo diagnosis, in vivo imaging, or therapy ofdiseases or conditions with distinctive antigens. The basic unit of anantibody structure is a complex of four polypeptides—two identical lowmolecular weight (“light”) chains and two identical high molecularweight (“heavy”) chains, linked together by both non-covalentassociations and by disulfide bonds. Different antibodies will haveanywhere from one to five of these basic units. The antibody may berepresented schematically as a “Y”. Each branch of the “Y” is formed bythe amino terminal portion of a heavy chain and an associated lightchain. The base of the “Y” is formed by the carboxy terminal portions ofthe two heavy chains. The node of the “Y” is referred to as the hingeregion.

Five human antibody classes (IgG, IgA, IgM, IgD and IgE), and withinthese classes, various subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1and IgA2) or subclass of immunoglobulin molecule., are recognized on thebasis of structural differences, such as the number of immunoglobulinunits in a single antibody molecule, the disulfide bridge structure ofthe individual units, and differences in chain length and sequence. Theclass and subclass of an antibody is its isotype.

The antibody can be an intact antibody or an antigen-binding antibodyfragment such as, for example, a Fab, a F(ab′), a F(ab′)₂, a Fd chain, asingle-chain Fv (scFv), a single-chain antibody, a disulfide-linked Fv(sdFv), a fragment comprising either a V_(L) or V_(H) domain, orfragments produced by a Fab expression library. Antigen-binding antibodyfragments, including single-chain antibodies, can comprise the variableregion(s) alone or in combination with the entirety or a portion of thefollowing: hinge region, C_(H)1, C_(H)2, C_(H)3, C_(H)4 and C_(L)domains. Also, antigen-binding fragments can comprise any combination ofvariable region(s) with a hinge region, C_(H)1, C_(H)2, C_(H)3, C_(H)4and C_(L) domains. In some embodiments, an antibody fragment comprisesat least one domain, or part of a domain, that includes interchaindisulfide bonds.

Typically, the antibodies are human, rodent (e.g., mouse and rat),donkey, sheep, rabbit, goat, guinea pig, camelid, horse, or chicken. Asused herein, “human” antibodies include antibodies having the amino acidsequence of a human immunoglobulin and include antibodies isolated fromhuman immunoglobulin libraries, from human B cells, or from animalstransgenic for one or more human immunoglobulin, as described infra and,for example in U.S. Pat. Nos. 5,939,598 and 6,111,166. The antibodiesmay be monospecific, bispecific, trispecific, or of greatermultispecificity.

In some embodiments, the constant domains have effector function. Theterm “antibody effector function(s),” or AEC, as used herein refers to afunction contributed by an Fc domain(s) of an Ig. Such function can beeffected by, for example, binding of an Fc effector domain(s) to an Fcreceptor on an immune cell with phagocytic or lytic activity or bybinding of an Fe effector domain(s) to components of the complementsystem. Typically, the effect(s) mediated by the Fe-binding cells orcomplement components result in inhibition and/or depletion of the CD70targeted cell. The effector function can be, for example,“antibody-dependent cellular cytotoxicity” or ADCC, “antibody-dependentcellular phagocytosis” or ADCP, “complement-dependent cytotoxicity” orCDC. In other embodiments, the constant domain lack one or more effectorfunctions.

The antibodies may be directed against antigen of interest, such asmedical and/or therapeutic interest. For example, the antigen can be oneassociated with pathogens (such as but not limited to viruses, bacteria,fungi, and protozoa), parasites, tumor cells, or particular medicalconditions. In the case of a tumor-associated antigen (TAA), the cancermay be of the immune system, lung, colon, rectum, breast, ovary,prostate gland, head, neck, bone, or any other anatomical location.Antigens of interest include, but are not limited to, CD30, CD40, LewisY, and CD70. In some embodiments, the antigen is CD2, CD20, CD22, CD33,CD38, CD40, CD52, HER2, EGFR, VEGF, CEA, HLA-DR, HLA-Dr10, CA125,CA15-3, CA19-9, L6, Lewis X, alpha fetoprotein, CA 242, placentalalkaline phosphatase, prostate specific antigen, prostatic acidphosphatase, epidermal growth factor, MAGE-1, MAGE-2, MAGE-3, MAGE-4,anti-transferrin receptor, p97, MUC1-KLH, gp100, MARTI, IL-2 receptor,human chorionic gonadotropin, mucin, P21, MPG, and Neu oncogene product.

Some specific useful antibodies include, but are not limited to, BR96mAb (Trail et al. (1993), Science 261:212-215), BR64 (Trail et al.(1997), Cancer Research 57:100-105), mAbs against the CD 40 antigen,such as S2C6 mAb (Francisco et al. (2000) Cancer Res. 60:3225-3231), andmAbs against the CD30 antigen, such as AC 10 (Bowen et al. (1993) J.Immunol. 151:5896-5906). Many other internalizing antibodies that bindto tumor specific antigens can be used, and have been reviewed (see,e.g., Franke et al. (2000), Cancer Biother Radiopharm. 15:459-76; Murray(2000), Semin Oncol. 27:64-70; Breitling et al., Recombinant Antibodies,John Wiley, and Sons, New York, 1998). The disclosures of thesereferences are incorporated by reference herein.

The term “tumor-specific antigen” as used herein will be understood toconnote an antigen characteristic of a particular tumor, or stronglycorrelated with such a tumor. However, tumor-specific antigens are notnecessarily unique to tumor tissue, however, i.e., that antibodies tothem may cross-react with antigens of normal tissue. Where atumor-specific antigen is not unique to tumor cells, it frequentlyoccurs that, as a practical matter, antibodies binding to tumor-specificantigens are sufficiently specific to tumor cells to carry out thedesired procedures without unwarranted risk or interference due tocross-reactions. Many factors contribute to this practical specificity.For example, the amount of antigen on the tumor cell may greatly exceedthe amount of the cross-reactive antigen found on normal cells, or theantigen on the tumor cells may be more effectively presented. Thereforethe term “tumor-specific antigen” relates herein to a specificity ofpractical utility, and is not intended to denote absolute specificity orto imply an antigen is unique to the tumor.

The antibody may be a polyclonal antibody or a monoclonal antibody. Whenthe subject is a human subject, the antibody may be obtained byimmunizing any animal capable of mounting a usable immune response tothe antigen. The animal may be a mouse, rat, goat, sheep, rabbit orother suitable experimental animal. The antigen may be presented in theform of a naturally occurring immunogen, or a synthetic immunogenicconjugate of a hapten and an immunogenic carrier. In the case of amonoclonal antibody, antibody producing cells of the immunized animalmay be fused with “immortal” or “immortalized” human or animal cells toobtain a hybridoma which produces the antibody. If desired, the genesencoding one or more of the immunoglobulin chains may be cloned so thatthe antibody may be produced in different host cells, and if desired,the genes may be mutated so as to alter the sequence and hence theimmunological characteristics of the antibody produced. Human monoclonalantibodies may be made by any of numerous techniques known in the art(e.g., Teng et al. (1983), Proc. Natl. Acad. Sci. USA. 80, 7308-7312;Kozbor et al. (1983) Immunology Today 4, 72-79; and Olsson et al.(1982), Meth. Enzymol. 92, 3-16).

The antibody can be, for example, a murine, a chimeric, humanized, orfully human antibody produced by techniques well-known to one of skillin the art. Recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are usefulantibodies. A chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine monoclonal and humanimmunoglobulin constant regions. (See, e.g., Cabilly et al., U.S. Pat.No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397, which areincorporated herein by reference in their entirety.) Humanizedantibodies are antibody molecules from non-human species having one ormore complementarity determining regions (CDRs) from the non-humanspecies and a framework region from a human immunoglobulin molecule.(See, e.g., Queen, U.S. Pat. No. 5,585,089, which is incorporated hereinby reference in its entirety.) Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart, for example using methods described in International PublicationNo. WO 87/02671; European Patent Publication No. 184,187; EuropeanPatent Publication No. 171496; European Patent Publication No. 173494;International Publication No. WO 86/01533; European Patent PublicationNo.12,023; Berter et al. (1988), Science 240:1041-1043; Liu et al.(1987), Proc. Natl. Mad. Sci. USA 84:3439-3443; Liu et al. (1987), J.Immunol. 139:3521-3526; Sun et al. (1987), Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al. (1987), Cancer. Res. 47:999-1005; Wood etal. (1985), Nature 314:446-449; and Shaw et al. (1988), J. Natl. CancerInst. 80:1553-1559; Morrison (1985), Science 229:1202-1207; Oi et al.(1986), BioTechniques 4:214; U.S. Pat. No. 5,225,539; Jones et al.(1986), Nature 321:552-525; Verhoeyan et al. (1988), Science 239:1534;and Beidler et al. (1988), J. Immunol. 141:4053-4060; each of which isincorporated herein by reference in its entirety.

Completely human antibodies can be produced, for example, usingtransgenic mice that are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. The transgenic mice are immunized in thenormal fashion with a selected antigen or a portion thereof. Monoclonalantibodies directed against the antigen can be obtained usingconventional hybridoma technology. The human immunoglobulin transgenesharbored by the transgenic mice rearrange during B cell differentiation,and subsequently undergo class switching and somatic mutation. Thus,using such a technique, it is possible to produce therapeutically usefulIgG, IgA, IgM and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg and Huszar (1995, Int. Rev.Immunol. 13:65-93). For a detailed discussion of this technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies, see, e.g., U.S. Pat. Nos. 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; each of which isincorporated herein by reference in its entirety. Other human antibodiescan be obtained commercially from, for example, Abgenix, Inc. (Freemont,Calif.) and Genpharm (San Jose, Calif.).

Completely human antibodies that recognize a selected epitope also canbe generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. See, e.g., Jespers et al. (1994),Biotechnology 12:899-903. Human antibodies can also be produced usingvarious techniques known in the art, including phage display libraries(Hoogenboom and Winter (1991), J. Mol. Biol. 227:381; Marks et al.(1991), J. Mol. Biol. 222:581; Quan and Carter (2002), “The rise ofmonoclonal antibodies as therapeutics.” In Anti-IgE and AllergicDisease, Jardieu, P. M. and Fick Jr., R. B, eds., Marcel Dekker, NewYork, N.Y., Chapter 20, pp. 427-469.

The antibody can also be a bispecific antibody. Methods for makingbispecific antibodies are known in the art. Traditional production offull-length bispecific antibodies is based on the coexpression of twoimmunoglobulin heavy chain-light chain pairs, where the two chains havedifferent specificities (Milstein et al., 1983, Nature 305:537-539).Because of the random assortment of immunoglobulin heavy and lightchains, these hybridomas (quadromas) produce a potential mixture of 10different antibody molecules, of which only one has the correctbispecific structure. Similar procedures are disclosed in InternationalPublication No. WO 93/08829, and in Traunecker et al. (1991), EMBO J.10:3655-3659.

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, C_(H)2, and C_(H)3 regions. It is preferred tohave the first heavy-chain constant region (C_(H)1) containing the sitenecessary for light chain binding, present in at least one of thefusions. Nucleic acids with sequences encoding the immunoglobulin heavychain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are co-transfected into asuitable host organism. This provides for great flexibility in adjustingthe mutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In an embodiment of this approach, the bispecific antibodies have ahybrid immunoglobulin heavy chain with a first binding specificity inone arm, and a hybrid immunoglobulin heavy chain-light chain pair(providing a second binding specificity) in the other arm. Thisasymmetric structure facilitates the separation of the desiredbispecific compound from unwanted immunoglobulin chain combinations, asthe presence of an immunoglobulin light chain in only one half of thebispecific molecule provides for a facile way of separation(International Publication No. WO 94/04690) which is incorporated hereinby reference in its entirety.

For further details for generating bispecific antibodies see, forexample, Suresh et al. (1986), Methods in Enzymology 121:210; Rodrigueset al. (1993), J. Immunology 151:6954-6961; Carter et al. (1992),Bio/Technology 10:163-167; Carter et al. (1995), J. of Hematotherapy4:463-470; Merchant et al. (1998), Nature Biotechnology 16:677-681.Using such techniques, bispecific antibodies can be prepared for use inthe treatment or prevention of disease.

Bifunctional antibodies are also described in European PatentPublication No. EPA 0 105 360. As disclosed in this reference, hybrid orbifunctional antibodies can be derived either biologically, i.e., bycell fusion techniques, or chemically, especially with cross-linkingagents or disulfide-bridge forming reagents, and may comprise wholeantibodies or fragments thereof. Methods for obtaining such hybridantibodies are disclosed for example, in International Publication WO83/03679, and European Patent Publication No. EPA 0 217 577, both ofwhich are incorporated herein by reference.

In other embodiments, the antibody is a fusion protein of an antibody,or a functionally active fragment thereof, for example in which theantibody is fused via a covalent bond (e.g., a peptide bond), at eitherthe N-terminus or the C-terminus to an amino acid sequence of anotherprotein (or portion thereof, preferably at least 10, 20 or 50 amino acidportion of the protein) that is not the antibody. Preferably, theantibody or fragment thereof is covalently linked to the other proteinat the N-terminus of the constant domain.

In yet other embodiments, the protein can be a fusion protein of thebinding portion of a non-antibody molecule fused via a covalent bond tothe antibody heavy and/or light chain constant region domain, optionallyincluding a hinge region. Such a fusion protein optionally can includeat least one, typically at least two, interchain disulfide bonds. Forexample, the fusion protein can include the C_(H)1 and C_(L) regions,and a hinge region.

III. Activation Methods

In general, a drug can be coupled to a protein or other suitablemolecule at an activatable site. Suitable activatable sites includeconjugation points such as thiol groups, amino groups (e.g., the epsilonamino group of lysine residues or at the N-terminus of proteins),vicinal hydroxyl groups (1,2-diols) (e.g., oxidized carbohydrates) andcarboxyl groups (e.g., the C-terminus of proteins, aspartic acid andglutamic acid residues, and carbohydrate, such as sialic acid residues).

A drug can be coupled directly to a conjugation point. For example, adrug can be attached by alkylation of the s-amino group of antibodylysines, reductive amination of oxidized carbohydrate or reaction with ahydrazide, transesterification between hydroxyl and carboxyl groups,amidation at amino groups or carboxyl groups, and conjugation to thiols(e.g., interchain thiols) or introduced thiols by, for example,alkylating lysines with 2-iminothiolane (Traut's reagent). Suitablemethods conjugating drugs to conjugation points are disclosed in, forexample, Current Protocols in Protein Science (John Wiley & Sons, Inc.),Chapter 15 (Chemical Modifications of Proteins) (the disclosure of whichis incorporated by reference herein in its entirety.)

A drug also can be coupled indirectly via another molecule, such as alinker. For example, a drug also can be conjugated via a maleimide groupcoupled to a sulthydryl group in, for example, but not limited to, thehinge region of an antibody. Antibody conjugates can be made by reactinga maleimide-derivatized form of the drug with the antibody. Morespecifically, antibody conjugates can be made by reducing an antibody toproduce the reduced antibody, producing an amine drug, derivatizing theamine drug with maleimide to produce a maleimide-derivatized drug, andreacting the maleimide-derivatized drug with the antibody.

In an exemplary embodiment, an IgG₁ such as cAC10 possesses manydisulfide bonds, only four of which are interchain. Because the fourinterchain disulfide bonds are clustered in the highly-flexible hingeregion and much more solvent-accessible than other (intra-chain)disulfide bonds, reduction with an excess of, for example, a reducingagent, such as but not limited to dithiothreitol (DTT),Tris(2-carboxyethyl)phosphine (TCEP), or 2-Mercaptoethanol, breaks allfour bonds and generates eight cysteines (i.e., containing the freethiol group). Conjugation of all eight cysteines with the drug-linkergenerates a fully-loaded conjugate with approximately eight drugs perantibody, as shown in see FIG. 7.

The present invention surprisingly demonstrates that the biologicalproperties of ADCs can be improved with antibodies having an average of2, 2.5, 4 or 6 drugs per antibody, which yields lower toxicity whilemaintaining the efficacy of fully loaded conjugates, i.e. conjugateshaving 8 drugs per antibody. The therapeutic window (concentration ofdrug-antibody conjugate where toxicity is first seen divided by thelowest efficacious dose) of partially-drug loaded conjugates is largerthan antibodies with 8 drugs. There are a number of ways to conjugatethe 8 cysteines with 4 drugs, yielding a large number of potential drugloaded species (9 total, with 0 through 8 drugs per antibody, see FIGS.1 and 7). For those antibodies that have 4 drugs, there are 6 possibleways to distribute the 4 drugs, yielding 6 isomers (See FIGS. 1 and 7).The homogeneity of the 8 drug loaded species is lost when 4 drugs perantibody is desired. For antibodies with 2 or 6 drugs per antibody,there are three possible ways to distribute the drugs on the molecules.

Methods to produce partially loaded ADCs (e.g., with 4 (E4) rather than8 drugs per antibody) include the following: method 1 (“partialreduction”) partial reduction of the antibody by a reducing agent suchas but not limited to DTT or TCEP followed by conjugation, and method 2(“full reduction and reoxidation”) full reduction of the antibody with areducing agent such as but not limited to DTT or TCEP, followed bypartial reoxidation of the antibody with a reoxidizing agent (forexample but not limited to 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB),4,4′-dithiodipyridine, 2,2′-dithiodipyridine, sodium tetrathionate, oriodosobenzoic acid) and finally conjugation. In the full reduction andreoxidation method, there are two aspects: 2a purification after DTNBreoxidation, and 2b, no purification after DTNB reoxidation (one potreoxidation and drug conjugation). These methods yield different percentof different species (e.g., for E4 from 25 to 40%) and also yielddifferent isomeric mixtures of the possible species. Encompassed in thedisclosure are hybrids and variations of the above methods which wouldbe known to one of skill in the art.

As an example for antibody drug conjugates, FIG. 1 shows the 6 possibleE4 species (referred to as 4A through 4F species) that can be generatedduring a conjugation reaction. Species 4A-D are not individuallydistinguishable by certain analytic methods; however, they can bedistinguished from both 4E and 4F.

In one embodiment of method 1 of partial reduction, conjugates with, forexample, 4 drugs per antibody can be made by full reduction of theantibody with, for example but not limited to DTT, to yield 8 antibodycysteines followed by conjugation to 4 equivalents of drug. This leadsto a mixture where antibodies have from 0 to 8 drugs. Alternatively, ifthe antibody is reduced by limiting quantities of, for example DTT, suchthat an average of only 2 of the 4 disulfides are reduced (liberating 4cysteines) followed by complete drug conjugation, only even drug loadedspecies (0, 2, 4, 6, and 8 drugs per antibody) will be formed. Thisreduces the complexity of the mixture, which can be further reduced bypurification to isolate these different drug loaded species.

In some embodiments, certain potential points of conjugation on aprotein can be selectively activated. This selective activation allowsfor ready assignability of the conjugation site(s) of a drug on theprotein. For example, treatment of an antibody (e.g., cAC10) withlimiting amounts of the strong reducing agents DTT or TCEP results inthe selective reduction of the heavy-light chain disulfides. In anotherexample, full DTT reduction of an antibody followed by partialreoxidation using a strong thiol oxidizing agent such as DTNB results inselective reoxidation of the heavy-light chain hinge disulfides, leadingto drug predominantly conjugated on the heavy chain in the hinge region.The isomer populations of E2 and E6 produced by both of these methodscan approach 90% isomeric homogeneity.

Following conjugation of the drug to the antibody, the conjugateddrug-antibody species can be separated. In some embodiments, theconjugated antibody species can be separated based on thecharacteristics of the antibody, the drug and/or the conjugate. Forexample, hydrophobic interaction chromatograph (HIC) has been successfulin isolating and separating species corresponding to 0, 2, 4, 6, and 8drugs per antibody. The yields of each of these drug loaded isomers bymethod 1 is close to what would be expected by a statisticaldistribution. The 4 drug loaded species is typically 30% of the totalmaterial.

Analytical methods have been developed to determine drug loading and thelocation of the drugs on the antibody (see also infra). Characterizationof the pure 4 drug loaded conjugates prepared by partial DTT reductionby Bioanalyzer (capillary electrophoresis) and HPLC on a crosslinkeddivinylbenzene column (PLRP) revealed that the drugs are predominantlylocated on cysteines that originally made disulfides between the heavyand light chains of the antibody. The specificity of the drug location,where one isomer is favored over the other five isomers, unlike theconvention, is unexpected.

In another embodiment, method 2, full reduction with partialreoxidation, to prepare drug-antibody conjugates with, for example, 4drugs, the antibody was fully reduced with, for example but not limitedto, DTT and then treated with limiting amounts of, for example but notlimited to, DTNB to reform some of the disulfides such that 4 antibodycysteine thiols remained. These cysteines were conjugated to drug andanalyzed by the methods described herein. The yield of 4 drug loadedantibodies in the mixture increased to as much as 40%, and once purifiedthe location of the drug favored placement on the cysteines thatoriginally made disulfides between the heavy chains in the hinge region.Both the yield of 4 drug loaded antibodies and the selectivity comparedto common convention, which favors a different isomer from certainpartial reduction methods, are unexpected. Using various chemical means,drug location within the antibody can be readily assigned for theproduction of different isomers.

If reduction is controlled by addition of limiting amounts of reducingagent, partial reduction occurs in which, on average, less than fourinter-chain disulfide bonds are broken per antibody. Because all fourinter-chain disulfide bonds are highly exposed, reduction proceedsthrough various pathways and produces partially-reduced antibodycomposed of a mixture of species with 0, 2, 4, 6, or 8 cysteines.Conjugation of partially-reduced antibody, therefore can generate amixture of conjugates with 0, 2, 4, 6, or 8 drugs per antibody, as shownin FIGS. 1 and 7. Depending on the extent of partial reduction, thedrug-load distribution (i.e., the percent of 0, 2, 4, 6, or 8drug-loaded species) changes.

Partial reduction not only produces a mixture containing species withvariable number of drugs per antibody, it also creates furtherheterogeneity as a result of the multiple locations of drug attachment.FIG. 7 shows that there is more than one isomer possible for the 2, 4,and 6 drug-loaded species.

Following conjugation of the drug to a protein, the conjugateddrug-protein species can be separated. For example, in some embodiments,the conjugated antibody species can be separated based on thecharacteristics of the antibody, the drug and/or the conjugate. Forexample, hydrophobic interaction chromatograph (HIC) has been successfulin isolating and separating species corresponding to 0, 2, 4, 6, and 8drugs per antibody.

IV. Analytical Methods

Various analytical methods can be used to determine the yields andisomeric mixtures of the conjugates. For example, in one embodiment HICis the analytical method used to determine yields and isomeric mixturesfrom resultant conjugates (e.g., for E4 conjugates). This technique isable to separate antibodies loaded with various numbers of drugs. Thedrug loading level can be determined based on the ratio of absorbances,e.g., at 250 nm and 280 nm. For example, if a drug can absorb at 250 nmwhile the antibody absorbs at 280 nm. The 250/280 ratio thereforeincreases with drug loading. Using the conjugation methods describedherein, generally antibodies with even numbers of drugs were observed tobe conjugated to the antibody since reduction of disulfides yields evennumbers of free cysteine thiols. FIGS. 2 and 3 show HIC separations forcAC10-vcMMAE produced by methods 2a and 2b, respectively. FIG. 4 showsthe percent composition for the various substitutions from thesechromatograms as well as from method 1. Method 1 yields about 30% E4,while method 2b yields about 40% E4.

HIC can also be used preparatively at milligram to grain levels topurify E4 from a mixture of substitution levels. Pure E4 from FIG. 3(collection time of 34-38 min indicated) was obtained and analyzed bytwo methods to determine the isomeric E4 mixture. First, an AgilentBioanalyzer was used, which denatures noncovalent interactions andseparates based on protein mass, yielding the following antibodycomponents in order of elution: light chain (L), heavy chain (H),heavy-light (HL), heavy-heavy (HH), heavy-heavy-light (HHL), andheavy-heavy-light-light (HHLL). The smaller species are formed whendisulfides are reduced and the free thiols conjugated to vcMMAE.

FIG. 1 also describes which antibody components will be observed fromdenaturation of the various E4 isomers. As can be seen in FIG. 5, pureE4 prepared by method 2b is dominated by HL, with a small amount of Land HHL. This result can be explained by the presence of mostly species4F (exclusively yields HL) with some species 4A-D (yielding HHL and L).Interestingly, the same analysis of cAC10-vcMMAE made by method 1 yieldsapproximately equal amounts of L, HL, and HH, which would be consistentwith a mixture of mostly species 4E and some species 4F.

Another embodiment of an analytical tool is chromatography on areversed-phase PLRP column; the column support is composed ofcrosslinked divinylbenzene, rather than a typical reversed phase columnbuilt on a silica support which can nonspecifically retain proteins.This denaturing and reductive technique cleanly separates the 6 speciesconsisting of light chain with 0 and 1 drug (L0 and L1) and heavy chainwith 0 through 3 drugs (HO through H3). FIG. 1 shows the drug loadinglevels that can be observed for the various E4 species. Pure E4 frommethod 2b was separated by PLRP in FIG. 6. Unmodified light chain (L0)and heavy chain with two drugs (H2) are the species expected from 4F,while L1 and H1 are expected from 4A-D. Together with the Bioanalyzer,these data are consistent with method 1 producing about a 2:1 mixture of4E to 4F while method 2b produces 2:1 4F to 4A-D. Thus using differentchemical conditions, both the E4 yield and distribution of E4 isomers issignificantly different between method 1 and 2b.

V. Compound Capable of Conjugation to Protein.

A protein may be conjugated with any drug of interest, including acytostatic agent or cytotoxic agent, an immunosuppressive agent, atoxin, a chelate, a compound, a molecule, a radionucleotide, or thelike.

Cytotoxic Agents and Cytostatic Agents

Cytotoxic and cytostatic drugs include antibiotics (e.g., adriamycin),antitumor agents such as auristatins and auristatin derivatives,methotrexate, mitomycin C, daunorubicin, doxorubicin, and vinblastine,5-fluorouracil DNA minor grove binders, DNA replication inhibitors,alkylating agents (e.g., platinum complexes such as cisplatin,mono(platinum), bis(platinum) and tri-nuclear platinum complexes andcarboplatin), antiparasitic agents (e.g., pentamidine isethionate),anthracyclines, antifolates, antimetabolites, chemotherapy sensitizers,duocarmycins, etoposides, fluorinated pyrimidines, ionophores,lexitropsins, nitrosoureas, platinols, pre-forming compounds, purineantimetabolites, puromycins, radiation sensitizers, steroids, taxanes,topoisomerase inhibitors, vinca alkaloids, antimicrobial agents,antimicrotubule agents, or the like. When an antibody is conjugated tosuch a drug, it serves to direct the drug to the sites where thecorresponding antigen occurs. Other agents and drugs which can becoupled to antibody are known, or can be easily ascertained, by those ofskill in the art.

Individual cytotoxic or cytostatic agents include, for example, anandrogen, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine,bleomycin, busulfan, buthionine sulfoximine, camptothecin, carboplatin,carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine,cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B,dacarbazine, dactinomycin (formerly actinomycin), daunorubicin,decarbazine, docetaxel, doxorubicin, an estrogen, 5-fluordeoxyuridine,5-fluorouracil, gramicidin D, hydroxyurea, idarubicin, ifosfamide,irinotecan, lomustine (CCNU), mechlorethamine, melphalan,6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone,nitroimidazole, paclitaxel, plicamycin, procarbizine, streptozotocin,tenoposide, 6-thioguanine, thioTEPA, topotecan, vinblastine,vincristine, vinorelbine, VP-16 and VM-26.

Other cytotoxic agents include, for example, dolastatins (see infra) DNAminor groove binders such as the enediynes (e.g., calicheamicin) andlexitropsins (see, also U.S. Pat. No. 6,130,237), duocarmycins, taxanes(e.g., paclitaxel and docetaxel), puromycins, CC-1065, SN-38, topotecan,morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin,echinomycin, combretastatin, netropsin, epothilone A, B or D,estramustine, cryptophysins, cemadotin, maytansinoids, discodermolide,eleutherobin, and mitoxantrone.

In certain embodiments, the cytotoxic agent is a chemotherapeutic suchas, for example, doxorubicin, melphalan, vinca alkaloids, methotrexate,mitomycin C or etoposide. In addition, potent agents such as CC-1065analogues, calicheamicin, maytansine, analogues of dolastatin 10,rhizoxin, and palytoxin can be linked to proteins.

In specific embodiments, the cytotoxic or cytostatic agent is auristatinE (also known in the art as dolastatin-10) or a derivative thereof.Typically, the auristatin E derivative is, e.g., an ester formed betweenauristatin E and a keto acid. For example, auristatin E can be reactedwith paraacetyl benzoic acid or benzoylvaleric acid to produce AEB andAEVB, respectively. Other typical auristatin derivatives include AFP,MMAF, and MMAE. The synthesis and structure of auristatin E and itsderivatives are described in U.S. patent application Ser. No. 09/845,786(U.S. Patent Application Publication No. 20030083263) and Ser. No.10/001,191; International Patent Application No. PCT/US03/24209:International Patent Application No. PCT/US02/13435: and U.S. Pat. Nos.6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149;5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191;5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278;4,816,444; and 4,486,414.

In certain embodiments, the cytotoxic or cytostatic agent is ananti-tubulin agent. Examples of anti-tubulin agents include, but are notlimited to, taxanes (e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)),T67 (Tularik), vinca alkyloids (e.g., vincristine, vinblastine,vindesine, and vinorelbine), and dolastatins (e.g., auristatin E, AFP,MMAF, MMAE, AEB, AEVB). Other antitubulin agents include, for example,baccatin derivatives, taxane analogs (e.g., epothilone A and B),nocodazole, colchicine and colcimid, estramustine, cryptophysins,cemadotin, maytansinoids, combretastatins, discodermolide, andeleutherobin.

In certain embodiments, the cytotoxic agent is a maytansinoid, anothergroup of anti-tubulin agents. For example, in specific embodiments, themaytansinoid is maytansine or DM-1 (ImmunoGen, Inc.; see also Chari etal. (1992), Cancer Res. 52:127-131).

In some embodiments, the therapeutic agent is not a radioisotope.

In some embodiments, the cytotoxic or immunosuppressive agent is anantimetabolite. The antimetabolite can be, for example, a purineantagonist (e.g., azothioprine or mycophenolate mofetil), adihydrofolate reductase inhibitor (e.g., methotrexate), acyclovir,gangcyclovir, zidovudine, vidarabine, ribavarin, azidothymidine,cytidine arabinoside, amantadine, dideoxyuridine, iododeoxyuridine,poscarnet, or trifluridine.

In other embodiments, the cytotoxic or immosuppressive agent istacrolimus, cyclosporine or rapamycin. In further embodiments, thecytotoxic agent is aldesleukin, alemtuzumab, alitretinoin, allopurinol,altretamine, amifostine, anastrozole, arsenic trioxide, bexarotene,bexarotene, calusterone, capecitabine, celecoxib, cladribine,Darbepoetin alfa, Denileukin diftitox, dexrazoxane, dromostanolonepropionate, epirubicin, Epoetin alfa, estramustine, exemestane.Filgrastim, floxuridine, fludarabine, fulvestrant, gemcitabine,goserelin, idarubicin, ifosfamide, imatinib mesylate, Interferonalfa-2a, irinotecan, letrozole, leucovorin, levamisole, meclorethamineor nitrogen mustard, megestrol, mesna, methotrexate, methoxsalen,mitomycin C, mitotane, nandrolone phenpropionate, oprelvekin,oxaliplatin, pamidronate, pegademase, pegaspargase, pegfilgrastim,pentostatin, pipobroman, plicamycin, porfimer sodium, procarbazine,quinacrine, rasburicase, Sargramostim, streptozocin, tamoxifen,temozolomide, teniposide, testolactone, thioguanine, toremifene,tretinoin, uracil mustard, vairubicin, vinblastine, vincristine,vinorelbine and zoledronate.

In some embodiments, the agent is an immunosuppressive agent. Theimmunosuppressive agent can be, for example, gancyclovir, tacrolimus,cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolatemofetil or methotrexate. Alternatively, the immunosuppressive agent canbe, for example, a glucocorticoid (e.g., cortisol or aldosterone) or aglucocorticoid analogue (e.g., prednisone or dexamethasone).

In some embodiments, the immunosuppressive agent is an anti-inflammatoryagent, such as arylcarboxylic derivatives, pyrazole-containingderivatives, oxicam derivatives and nicotinic acid derivatives. Classesof anti-inflammatory agents include, for example, cyclooxygenaseinhibitors, 5-lipoxygenase inhibitors, and leukotriene receptorantagonists.

Suitable cyclooxygenase inhibitors include meclofenamic acid, mefenamicacid, carprofen, diclofenac, diflunisal, fenbufen, fenoprofen,ibuprofen, indomethacin, ketoprofen, nabumetone, naproxen, sulindac,tenoxicam, tolmetin, and acetylsalicylic acid.

Suitable lipoxygenase inhibitors include redox inhibitors (e.g.,catechol butane derivatives, nordihydroguaiaretic acid (NDGA),masoprocol, phenidone, Ianopalen, indazolinones, naphazatrom,benzofuranol, alkylhydroxylamine), and non-redox inhibitors (e.g.,hydroxythiazoles, methoxyalkylthiaiazoles, benzopyrans and derivativesthereof, methoxytetrahydropyran, boswellic acids and acetylatedderivatives of boswellic acids, and quinolinemethoxyphenylacetic acidssubstituted with cycloalkyl radicals), and precursors of redoxinhibitors.

Other suitable lipoxygenase inhibitors include antioxidants (e.g.,phenols, propyl gallate, flavonoids and/or naturally occurringsubstrates containing flavonoids, hydroxylated derivatives of theflavones, flavonol, dihydroquercetin, luteolin, galangin, orobol,derivatives of chalcone, 4,2′,4′-trihydroxychalcone, ortho-aminophenols,N-hydroxyureas, benzofuranols, ebselen and species that increase theactivity of the reducing selenoenzymes), iron chelating agents (e.g.,hydroxamic acids and derivatives thereof, N-hydroxyureas,2-benzyl-1-naphthol, catechols, hydroxylamines, carnosol trolox C,catechol, naphthol, sulfasalazine, zyleuton, 5-hydroxyanthranilic acidand 4-(omega-arylalkyl)phenylalkanoic acids), imidazole-containingcompounds (e.g., ketoconazole and itraconazole), phenothiazines, andbenzopyran derivatives.

Yet other suitable lipoxygenase inhibitors include inhibitors ofeicosanoids (e.g., octadecatetraenoic, eicosatetraenoic,docosapentaenoic, eicosahexaenoic and docosahexaenoic acids and estersthereof, PGE1 (prostaglandin E1), PGA2 (prostaglandin A2), viprostol,15-monohydroxyeicosatetraenoic, 15-monohydroxy-eicosatrienoic and15-monohydroxyeicosapentaenoic acids, and leukotrienes B5, C5 and D5),compounds interfering with calcium flows, phenothiazines,diphenylbutylamines, verapamil, fuscoside, curcumin, chlorogenic acid,caffeic acid, 5,8,11,14-eicosatetrayenoic acid (ETYA),hydroxyphenylretinamide, lonapalen, esculin, diethylcarbamazine,phenantroline, baicalein, proxicromil, thioethers, diallyl sulfide anddi-(1-propenyl) sulfide.

Leukotriene receptor antagonists include calcitriol, ontazolast, BayerBay-x-1005, Ciba-Geigy CGS-25019C, ebselen, Leo Denmark ETH-615, LillyLY-293111, Ono ONO-4057, Terumo TMK-688, Boehringer Ingleheim BI-RM-270,Lilly LY 213024, Lilly LY 264086, Lilly LY 292728, Ono ONO LB457, Pfizer105696, Perdue Frederick PF 10042, Rhone-Poulenc Rorer RP 66153,SmithKline Beecham SB-201146, SmithKline Beecham SB-201993, SmithKlineBeecham SB-209247, Searle SC-53228, Sumitamo SM 15178, American HomeProducts WAY 121006, Bayer Bay-o-8276, Warner-Lambert CI-987,Warner-Lambert CI-987BPC-15LY 223982, Lilly LY 233569, Lilly LY-255283,MacroNex MNX-160, Merck and Co. MK-591, Merck and Co. MK-886, OnoONO-LB-448, Purdue Frederick PF-5901, Rhone-Poulenc Rorer RG 14893,Rhone-Poulenc Rorer RP 66364, Rhone-Poulenc Rorer RP 69698, ShionoogiS-2474, Searle SC-41930, Searle SC-50505, Searle SC-51146, SearleSC-52798, SmithKline Beecham SK&F-104493, Leo Denmark SR-2566, TanabeT-757 and Teijin TEI-1338.

Toxins

Toxins are usefully conjugated to antibodies specific for antigensassociated with tumor, parasite or microbial cells. The toxin may befrom, e.g., a plant (e.g., ricin or abrin), animal (e.g., a snakevenom), or microbial (e.g., diphtheria or tetanus toxin).

Besides antibodies, the drugs or toxins may be conjugated to othercarrier proteins, such as albumin.

Conjugation of Drugs to Protein

The drug has, or is modified to include, a group reactive with aconjugation point on the protein. For example, a drug can be attached byalkylation (e.g., at the ε-amino group of antibody lysines or theN-terminus of protein), reductive amination of oxidized carbohydrate,transesterification between hydroxyl and carboxyl groups, amidation atamino groups or carboxyl groups, and conjugation to thiols. For aexamples of chemistries that can be used for conjugation, see, e.g.,Current Protocols in Protein Science (John Wiley & Sons, Inc.), Chapter15 (Chemical Modifications of Proteins) (the disclosure of which isincorporated by reference herein in its entirety.)

For example, when chemical activation of the protein results information of free thiol groups, the protein may be conjugated with asulfhydryl reactive agent. In one aspect, the agent is one which issubstantially specific for free thiol groups. Such agents include, forexample, malemide, haloacetamides (e.g., iodo, bromo or chloro),haloesters (e.g., iodo, bromo or chloro), halomethyl ketones (e.g.,iodo, bromo or chloro), benzylic halides (e.g., iodide, bromide orchloride), vinyl sulfone and pyridyithio.

Sulfhydryl Reactive Agents

Sulfyhydryl reactive agents include alpha-haloacetyl compounds such asiodoacetamide, maleimides such as N-ethylmaleimide, mercury derivativessuch as 3,6-bis-(mercurimethyl)dioxane with counter ions of acetate,chloride or nitrate, and disulfide derivatives such as disulfide dioxidederivatives, polymethylene bismethane thiosulfonate reagents andcrabescein (a fluorescent derivative of fluorescein containing two freesulfhydryl groups which have been shown to add across disulfide bonds ofreduced antibody).

Alpha-haloacetyl compounds such as iodoacetate readily react withsulfhydryl groups to form amides. These compounds have been used tocarboxymethylate free thiols. They are not strictly SH specific and willreact with amines. The reaction involves nucleophilic attack of thethiolate ion resulting in a displacement of the halide. The reactivehaloacetyl moiety, X—CH₂ CO—, has been incorporated into compounds forvarious purposes. For example, bromotrifluoroacetone has been used forF-19 incorporation, and N-chloroacetyliodotyramine has been employed forthe introduction of radioactive iodine into proteins.

Maleimides such as N-ethylmaleimide are considered to be fairly specificto sulfhydryl groups, especially at pH values below 7, where othergroups are protonated. Thiols undergo Michael reactions with maleimidesto yield exclusively the adduct to the double bond. The resultingthioether bond is very stable. They also react at a much slower ratewith amino and imidazoyl groups. At pH 7, for example, the reaction withsimple thiols is about 1,000 fold faster than with the correspondingamines. The characteristic absorbance change in the 300 nm regionassociated with the reaction provides a convenient method for monitoringthe reaction. These compounds are stable at low pH but are susceptibleto hydrolysis at high pH. See generally Wong, Chemistry of ProteinConjugation and Cross-linking; CRC Press, Inc., Boca Raton, 1991:Chapters 2 and 4).

A molecule (such as a drug) which is not inherently reactive withsulfhydryls may still be conjugated to the chemically activated proteinsby means of a bifunctional crosslinking agent which bears both a groupreactive with the molecule of interest and a sulfhydryl reactive group.This agent may be reacted simultaneously with both the molecule ofinterest (e.g., through an amino, carboxy or hydroxy group) and thechemically activated protein, or it may be used to derivatize themolecule of interest to form a partner molecule which is then sulfhydrylreactive by virtue of a moiety derived from the agent, or it may be usedto derivatize the chemically activated protein to make it reactive withthe molecule of interest.

Linkers

The drug can be linked to a protein by a linker. Suitable linkersinclude, for example, cleavable and non-cleavable linkers. A cleavablelinker is typically susceptible to cleavage under intracellularconditions. Suitable cleavable linkers include, for example, a peptidelinker cleavable by an intracellular protease, such as lysosomalprotease or an endosomal protease. In exemplary embodiments, the linkercan be a dipeptide linker, such as a valine-citrulline (val-cit) or aphenylalanine-lysine (phe-lys) linker. Other suitable linkers includelinkers hydrolyzable at a pH of less than 5.5, such as a hydrazonelinker. Additional suitable cleavable linkers include disulfide linkers.

A linker can include a group for linkage to the protein. For example,linker can include an amino, hydroxyl, carboxyl or sulfhydryl reactivegroups (e.g., malemide, haloacetamides (e.g., iodo, bromo or chloro),haloesters (e.g., iodo, bromo or chloro), halomethyl ketones (e.g.,iodo, bromo or chloro), benzylic halides (e.g., iodide, bromide orchloride), vinyl sulfone and pyridyithio). See generally Wong, Chemistryof Protein Conjugation and Cross-linking; CRC Press, Inc., Boca Raton,1991.

In certain embodiments, the antibody or protein drug conjugate can be ofthe following formula:

or pharmaceutically acceptable salts or solvates thereof.

wherein:

Ab is an antibody or other protein,

A is a stretcher unit,

a is 0 or 1,

each W is independently a linker unit,

w is an integer ranging from 0 to 12,

Y is a spacer unit, and

y is 0, 1 or 2,

p ranges from 1 to about 20, and

D is a drug, a label or other molecule.

z is the number of potential conjugation sites on the protein, whereinp<z.

In other embodiments, p can be, for example, 2, 4, 8, 10, 12, 16, 25, ormore.

A stretcher unit can is capable of linking a linker unit to an antibodyor other protein. The stretcher unit has a functional group that canform a bond with a functional group of the antibody or other protein.Useful functional groups include, but are not limited to, sulfhydryl(—SH), amino, hydroxyl, carboxy, the anomeric hydroxyl group of acarbohydrate, and carboxyl.

The linker unit is typically an amino acid unit, such as for example adipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide,heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide ordodecapeptide unit. The linker unit can be cleavage or non-cleavableinside the cell.

A spacer unit, if present, links a linker unit to the drug. alternately,a spacer unit can link a stretcher unit to a drug moiety when the linkerunit is absent. The spacer unit can also link a drug to an antibody orprotein when both the linker unit and stretcher unit are absent.

VI. Conjugates and Their Uses

In vitro Immunodiagnosis.

In one embodiment, a protein (e.g., an antibody) is conjugated to adetectable label for use in in vitro immunodiagnosis. The label may be aradiolabel, fluorophore, or enzyme which is directly or indirectlyconjugatable to conjugation point (e.g., a free thiol group) of thechemically activated antibody. The sample may be of clinical (e.g.,blood, urine, semen, or cerebrospinal fluid, or a solid tissue or organ)or non-clinical (e.g., soil, water, food) nature. The assay may bequalitative or quantitative, and in any desired format, includingsandwich and competitive formats. Numerous immunoassay formats, labels,immobilization techniques, etc., are disclosed in the followingpublications, hereby incorporated by reference herein: O'Sullivan(1976), Annals Clin. Biochem. 16:221-240; McLaren (1981), Med. Lab. Sci.38:245-51; Ollerich (1984), J. Clin. Chem. Clin. Biochem. 22:895-904;Ngo and Lenhoff (1982), Mol. Cell. Biochem., 44:3-12.

Immunoimaging.

An immunoconjugate may also be used for in vivo immunoimaging. For thispurpose, the protein (e.g., an antibody) is labeled by means whichpermit external visualization of its position or location within asubject or part thereof, such as an organ. Typically, an immunoimagingagent will be an antibody labeled directly (as with Technetium) orindirectly (as with chelated Indium) with a suitable radioisotope. Afterinjection into the patient, the location of the conjugate may be trackedby a detector sensitive to particles emitted by the radiolabel, e.g., agamma-scintillation camera in the case of a gamma emitter.

Immunotherapy.

For immunotherapy, a protein can be conjugated to suitable drug, such asa cytotoxic or cytostatic agent, an immunosuppressive agent, aradioisotope, a toxin, or the like. The conjugate can be used forinhibiting the multiplication of a tumor cell or cancer cell, causingapoptosis in a tumor or cancer cell, or for treating cancer in apatient. The conjugate can be used accordingly in a variety of settingsfor the treatment of animal cancers. The conjugate can be used todeliver a drug to a tumor cell or cancer cell. Without being bound bytheory, in some embodiments, the conjugate binds to or associates with acancer-cell or a tumor-associated antigen, and the conjugate and/or drugcan be taken up inside a tumor cell or cancer cell throughreceptor-mediated endocytosis. The antigen can be attached to a tumorcell or cancer cell or can be an extracellular matrix protein associatedwith the tumor cell or cancer cell. Once inside the cell, one or morespecific peptide sequences within the conjugte (e.g., in a linker) arehydrolytically cleaved by one or more tumor-cell orcancer-cell-associated proteases, resulting in release of the drug. Thereleased drug is then free to migrate within the cell and inducecytotoxic or cytostatic or other activities. In some embodiments, thedrug is cleaved from the antibody outside the tumor cell or cancer cell,and the drug subsequently penetrates the cell, or acts at the cellsurface.

Thus, in some embodiments, the conjugate or other protein binds to thetumor cell or cancer cell. In some embodiments, the conjugate binds to atumor cell or cancer cell antigen which is on the surface of the tumorcell or cancer cell. In other embodiments, the conjugate binds to atumor cell or cancer cell antigen which is an extracellular matrixprotein associated with the tumor cell or cancer cell.

The specificity of the protein for a particular tumor cell or cancercell can be important for determining those tumors or cancers that aremost effectively treated. For example, antibodies having an anti-CD30 oran anti-CD40 antibody or other binding protein can be useful fortreating hematologic malignancies.

Other particular types of cancers that can be treated with theprotein-drug conjugates include, but are not limited to, solid tumors,including but not limited to: fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer,pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostatecancer, esophogeal cancer, stomach cancer, oral cancer, nasal cancer,throat cancer, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicularcancer, small cell lung carcinoma, bladder carcinoma, lung cancer,epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skincancer, melanoma, neuroblastoma, retinoblastoma, blood-borne cancers(including but not limited to: acute lymphoblastic leukemia “ALL”, acutelymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia,acute myeloblastic leukemia “AML”, acute promyelocytic leukemia “APL”,acute monoblastic leukemia, acute erythroleukemic leukemia, acutemegakaryoblastic leukemia, acute myelomonocytic leukemia, acutenonlymphocyctic leukemia, acute undifferentiated leukemia, chronicmyelocytic leukemia “CML”, chronic lymphocytic leukemia “CLL”, hairycell leukemia, multiple myeloma), acute and chronic leukemias (e.g.,lymphoblastic, myelogenous, lymphocytic, and myelocytic leukemias), andLymphomas (e.g., Hodgkin's disease, non-Hodgkin's Lymphoma, Multiplemyeloma, Waldenstrom's macroglobulinemia, Heavy chain disease, andPolycythemia vera). The proteins provide conjugation-specific tumor orcancer targeting.

Multi-Modality Therapy For Cancer

As discussed above, cancers, including, but not limited to, a tumor,metastasis, or other disease or disorder characterized by uncontrolledcell growth, can be treated or prevented by administration of aprotein-drug conjugate.

In other embodiments, methods for treating or preventing cancer areprovided, including administering to a patient in need thereof aneffective amount of a conjugate and a chemotherapeutic agent. In someembodiments, the chemotherapeutic agent is that with which treatment ofthe cancer has not been found to be refractory. In some embodiments, thechemotherapeutic agent is that with which the treatment of cancer hasbeen found to be refractory. The conjugate can be administered to apatient that has also undergone an treatment, such as surgery fortreatment for the cancer. In another embodiment, the additional methodof treatment is radiation therapy.

In an exemplary embodiment, the protein-drug conjugate is administeredconcurrently with the chemotherapeutic agent or with radiation therapy.In another exemplary embodiment, the chemotherapeutic agent or radiationtherapy is administered prior or subsequent to administration of theprotein-drug conjugate, in one aspect at least an hour, five hours, 12hours, a day, a week, a month, in further aspects several months (e.g.,up to three months), prior or subsequent to administration of theconjugate.

A chemotherapeutic agent can be administered over a series of sessions.Any one or a combination of the chemotherapeutic agents listed below canbe administered. With respect to radiation, any radiation therapyprotocol can be used depending upon the type of cancer to be treated.For example, but not by way of limitation, x-ray radiation can beadministered; in particular, high-energy megavoltage (radiation ofgreater that 1 MeV energy) can be used for deep tumors, and electronbeam and orthovoltage x-ray radiation can be used for skin cancers.Gamma-ray emitting radioisotopes, such as radioactive isotopes ofradium, cobalt and other elements, can also be administered.

Additionally, methods of treatment of cancer with a protein-drugconjugate are provided as an alternative to chemotherapy or radiationtherapy, where the chemotherapy or the radiation therapy has proven orcan prove too toxic, e.g., results in unacceptable or unbearable sideeffects, for the subject being treated. The animal being treated can,optionally, be treated with another cancer treatment such as surgery,radiation therapy or chemotherapy, depending on which treatment is foundto be acceptable or bearable.

The protein-drug conjugate can also be used in an in vitro or ex vivofashion, such as for the treatment of certain cancers, including, butnot limited to leukemias and lymphomas, such treatment involvingautologous stem cell transplants. This can involve a multi-step processin which the animal's autologous hematopoietic stem cells are harvestedand purged of all cancer cells, the animal's remaining bone-marrow cellpopulation is then eradicated via the administration of a high dose of aconjugate with or without accompanying high dose radiation therapy, andthe stem cell graft is infused back into the animal. Supportive care isthen provided while bone marrow function is restored and the animalrecovers.

Multi-Drug Therapy For Cancer

Methods for treating cancer include administering to a patient in needthereof an effective amount of an a protein-drug conjugate and anothertherapeutic agent that is an anti-cancer agent are disclosed. Suitableanticancer agents include, but are not limited to, methotrexate, taxol,L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine,cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin,mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustards,cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecins,bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin,plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine,vinorelbine, paclitaxel, and docetaxel.

The anti-cancer agent includes, but is not limited to, a drug such as analkylating agents such as a nitrogen mustard (e.g., cyclophosphamide,ifosfamide, trofosfamide, chlorambucil, melphalan), nitrosoureas (e.g.,carmustine (BCNU), lomustine (CCNU)), alkylsulphonates (e.g., busulfan,treosulfan), triazenes (e.g., decarbazine), Platinum containingcompounds (e.g., cisplatin, carboplatin); plant alkaloids, such as vincaalkaloids (e.g., vincristine, vinblastine, vindesine, vinorelbine),taxoids (e.g., paclitaxel, docetaxol); DNA topoisomerase inhibitors suchas epipodophyllins (e.g., etoposide, teniposide, topotecan,9-aminocamptothecin, camptothecin, crisnatol, mitomycins (e.g.,mitomycin C); anti-metabolites such as anti-folates such as DHFRinhibitors (e.g., methotrexate, trimetrexate), IMP dehydrogenaseinhibitors (mycophenolic acid, tiazofurin, ribavirin, EICAR) andribonucleotide reductase inhibitors (e.g., hydroxyurea, deferoxamine),pyrimidine analogs such as uracil analogs (5-fluorouracil, floxuridine,doxifluridine, ratitrexed), cytosine analogs (e.g., cytarabine (ara C),cytosine arabinoside, fludarabine), and purine analogs (e.g.,mercaptopurine, thioguanine); hormonal therapies, such as receptorantagonists, such as anti-estrogens (e.g., tamoxifen, raloxifene,megestrol), LHRH agonists (e.g., goscrclin, leuprolide acetate), andanti-androgens (e.g., flutamide, bicalutamide; retinoids/deltoids suchas vitamin D3 analogs (e.g., EB 1089, CB 1093, KH 1060), photodynamictherapies (e.g., vertoporfin (BPD-MA), phthalocyanine, photosensitizerPc4, demethoxy-hypocrellin A (2BA-2-DMHA)), cytokines (e.g,interferon-α, interferon-γ, tumor necrosis factor), as well as otherdrugs, such as gemcitabine, velcade, revamid, thalamid, isoprenylationinhibitors (e.g., lovastatin), dopaminergic neurotoxins (e.g.,1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g.,staurosporine), actinomycin (e.g., actinomycin D, dactinomycin),bleomycin, bleomycin A2, bleomycin B2, peplomycin), anthracyclines(daunorubicin, Doxorubicin (adriamycin), idarubicin, epirubicin,pirarubicin, zorubicin, mtoxantrone), MDR inhibitors (e.g., verapamil),and Ca²⁺ ATPase inhibitors (e.g., thapsigargin)

Treatment Of Autoimmune Diseases

The protein-drug conjugates are useful for killing or inhibiting thereplication of a cell that produces an autoimmune disease or fortreating an autoimmune disease. The conjugates can be used accordinglyin a variety of settings for the treatment of an autoimmune disease in apatient. The conjugates can be used to deliver a drug to a target cell.Without being bound by theory, in one embodiment, the conjugatesassociate with an antigen on the surface of a target cell, and theconjugate is then taken up inside a target-cell throughreceptor-mediated endocytosis. Once inside the cell, one or morespecific peptide sequences (e.g., within a linker) are enzymatically orhydrolytically cleaved, resulting in release of a drug. The releaseddrug is then free to migrate in the cytosol and induce cytotoxic orcytostatic activities. In an alternative embodiment, the drug is cleavedfrom the conjugate outside the target cell, and the drug subsequentlypenetrates the cell.

In some embodiments, the protein-drug conjugate binds to an autoimmuneantigen. In one aspect, the antigen is on the surface of a cell involvedin an autoimmune condition. In some embodiments, an antibody binds to anautoimmune antigen which is on the surface of a cell. In an exemplaryembodiment, an antibody binds to activated lymphocytes that areassociated with the autoimmune disease state. In a further embodiment,the conjugates kill or inhibit the multiplication of cells that producean autoimmune antibody associated with a particular autoimmune disease.

Particular types of autoimmune diseases that can be treated with theprotein-drug conjugate include, but are not limited to, Th2 lymphocyterelated disorders (e.g., atopic dermatitis, atopic asthma,rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemicsclerosis, and graft versus host disease); Th1 lymphocyte relateddisorders (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis,Sjorgren's syndrome, Hashimoto's thyroiditis, Grave's disease, primarybiliary cirrhosis, Wegener's granulomatosis and tuberculosis); andactivated B lymphocyte related disorders (e.g., systemic lupuserythematosus, Goodpasture's syndrome, rheumatoid arthritis and type Idiabetes). Other autoimmune diseases include, but are not limited to,active chronic hepatitis, Addison's disease, allergic alveolitis,allergic reaction, allergic rhinitis, Alport's Syndrome, anaphlaxis,ankylosing spondylitis, anti-phosholipid syndrome, arthritis,ascariasis, aspergillosis, atopic allergy, atropic dermatitis, atropicrhinitis, Behcet's disease, Bird-Fancier's Lung, bronchial asthma,Caplan's syndrome, cardiomyopathy, Celiac disease, Chagas' disease,chronic glomerulonephritis, Cogan's Syndrome, cold agglutinin disease,congenital rubella infection, CREST syndrome, Crohn's disease,cryoglobulinemia, Cushing's syndrome, dermatomyositis, discoid lupus,Dressler's syndrome, Eaton-Lambert syndrome, echovirus infection,encephalomyelitis, endocrine opthalmopathy, Epstein-Barr virusinfection, equine heaves, erythematosis, Evan's syndrome, Felty'ssyndrome, fibromyalgia, Fuch's cyclitis, gastric atrophy,gastrointestinal allergy, giant cell arteritis, glomerulonephritis,goodpasture's syndrome, graft v. host disease, Graves' disease,Guillain-Barre disease, Hashimoto's thyroiditis, hemolytic anemia,Henoch-Schonlein Purpura, idiopathic adrenal atrophy, idiopathicpulmonary fibritis, IgA nephropathy, inflammatory bowel disease,insulin-dependent diabetes mellitus, juvenile arthritis, juvenilediabetes mellitus (Type I), Lambert-Eaton syndrome, laminitis, lichenplanus, lupoid hepatitis, lupus, lymphopenia, Meniere's disease, mixedconnective tissue disease, multiple sclerosis, myasthenia gravis,pernicious anemia, polyglandular syndromes, presenile dementia, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Raynauds phenomenon, recurrent abortion, Reiter's syndrome,rheumatic fever, rheumatoid arthritis, Sampter's syndrome,schistosomiasis, Schmidt's syndrome, scleroderma, Shulman's syndrome,Sjorgen's syndrome, stiff-man syndrome, sympathetic ophthalmia, systemiclupus erythematosis, Takayasu's arteritis, temporal arteritis,thyroiditis, thrombocytopenia, thyrotoxicosis, toxic epidermalnecrolysis, Type B insulin resistance, Type I diabetes mellitus,ulcerative colitis, uveitis, vitiligo, Waldenstrom's macroglobulemia,and Wegener's granulomatosis.

Multi-Drug Therapy of Autoimmune Diseases

Methods for treating an autoimmune disease are also disclosed thatinclude administering to a patient in need thereof an effective amountof a protein-drug conjugate alone or in combination another therapeuticagent known for the treatment of an autoimmune disease. Theanti-autoimmune disease agent can include, but is not limited to, thefollowing: cyclosporine, cyclosporine A, mycophenylate mofetil,sirolimus, tacrolimus, enanercept, prednisone, azathioprine,methotrexate, cyclophosphamide, aminocaproic acid, chloroquine,hydroxychloroquine, hydrocortisone, dexamethasone, chlorambucil, DHEA,danazol, bromocriptine, meloxicam and infliximab.

Treatment of Infectious Diseases

The protein-drug conjugates are useful for killing or inhibiting themultiplication of a cell that produces an infectious disease or fortreating an infectious disease. The conjugates can be used accordinglyin a variety of settings for the treatment of an infectious disease in apatient. The ADCs can be used to deliver a drug to a target cell. In oneembodiment, the antibody binds to the infectious disease cell. In someembodiments, the conjugate kills or inhibit the multiplication of cellsthat produce a particular infectious disease. Particular types ofinfectious diseases that can be treated with the conjugates include, butare not limited to, the following: bacterial diseases, such asdiphtheria, pertussis, occult bacteremia, urinary tract infection,gastroenteritis, cellulites, epiglottitis, tracheitis, adenoidhypertrophy, retropharyngeal abcess, impetigo, ecthyma, pneumonia,endocarditis, septic arthritis, pneumococcal, peritonitis, bactermia,meningitis, acute purulent meningitis, urethritis, cervicitis,proctitis, pharyngitis, salpingitis, epididymitis, gonorrhea, syphilis,listeriosis, anthrax, nocardiosis, salmonella, typhoid fever, dysentery,conjunctivitis, sinusitis, brucellosis, tularemia, cholera, bubonicplague, tetanus, necrotizing enteritis, and actinomycosis; mixedanaerobic infections, such as syphilis, relapsing fever, leptospirosis,Lyme disease, rat bite fever, tuberculosis, lymphadenitis, leprosy,chlamydia, chlamydial pneumonia, trachoma, and inclusion conjunctivitis;systemic fungal diseases such as histoplamosis, coccidiodomycosis,blastomycosis, sporotrichosis, cryptococcsis, systemic candidiasis,aspergillosis, mucormycosis, mycetoma, and chromomycosis; rickettsialdiseases such as typhus, Rocky Mountain Spotted Fever, ehrlichiosis,Eastern Tick-Borne Rickettsioses, rickettsialpox, Q fever andbartonellosis; parasitic diseases such as malaria, babesiosis, Africansleeping sickness, Chagas' disease, leishmaniasis, Dum-Dum fever,toxoplasmosis, meningoencephalitis, keratitis, entamebiasis, giardiasis,cryptosporidiosis, isosporiasis, cyclosporiasis, microsporidiosis,ascariasis, whipworm infection, hookworm infection, threadworminfection, ocular larva migrans, trichinosis, Guinea worm disease,lymphatic Filariasis, loiasis, River Blindness, canine heartwouninfection, schistosomiasis, swimmer's itch, Oriental lung fluke,Oriental liver fluke, fascioliasis, fasciolopsiasis, opisthorchiasis,tapeworm infections, hydatid disease, and alveolar hydatid disease;viral diseases such as measles, subacute sclerosing panencephalitis,common cold, mumps, rubella, roseola, Fifth Disease, chickenpox,respiratory syncytial virus infection, croup, bronchiolitis, infectiousmononucleosis, poliomyelitis, herpangina, hand-foot-and-mouth disease,Bornholm disease, genital herpes, genital warts, aseptic meningitis,myocarditis, pericarditis, gastroenteritis, acquired immunodeficiencysyndrome (AIDS), human immunodeficiency virus (HIV), Reye's syndrome,Kawasaki syndrome, influenza, bronchitis, viral “Walking” pneumonia,acute febrile respiratory disease, acute pharyngoconjunctival fever,epidemic keratoconjunctivitis, Herpes Simplex Virus 1 (HSV-1), HerpesSimplex Virus 2 (HSV-2), shingles, cytomegalic inclusion disease,rabies, progressive multifocal leukoencephalopathy, kuru, fatal familialinsomnia, Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinkerdisease, tropical spastic paraparesis, western equine encephalitis,California encephalitis, St. Louis encephalitis, Yellow Fever, Dengue,lymphocytic choriomeningitis, Lassa fever, hemorrhagic fever, Hantviruspulmonary syndrome, Marburg virus infections, Ebola virus infections andsmallpox.

Multi-Drug Therapy of Infectious Diseases

Methods for treating an infectious disease are disclosed as includingadministering to a patient in need thereof a protein-drug conjugatealone or in combination with another therapeutic agent that is ananti-infectious disease agent. The anti-infectious disease agent can be,but not limited to, the following: β-lactam antibiotics, such aspenicillin G, penicillin V, cloxacilliin, dicloxacillin, methicillin,nafcillin, oxacillin, ampicillin, amoxicillin, bacampicillin,azlocillin, carbenicillin, mezlocillin, piperacillin and ticarcillin;aminoglycosides such as amikacin, gentamicin, kanamycin, neomycin,netihnicin, streptomycin and tobramycin; macrolides such asazithromycin, clarithromycin, erythromycin, lincomycin and clindamycin;tetracyclines such as demeclocycline, doxycycline, minocycline,oxytetracycline and tetracycline; quinolones such as cinoxacin, andnalidixic acid; fluoroquinolones such as ciprofloxacin, enoxacin,grepafloxacin, levofloxacin, lomefloxacin, norfloxacin, ofloxacin,sparfloxacin and trovafloxicin; polypeptides such as bacitracin,colistin and polymyxin B; sulfonamides such as sulfisoxazole,sulfamethoxazole, sulfadiazine, sulfamethizole and sulfacetamide; andother antibacterial agents, such as trimethoprim, sulfamethazole,chloramphenicol, vancomycin, metronidazole, quinupristin, dalfopristin,rifampin, spectinomycin and nitrofurantoin; and antiviral agents, suchas general antiviral agents such as idoxuradine, vidarabine,trifluridine, acyclovir, famcicyclovir, pencicyclovir, valacyclovir,gancicyclovir, foscarnet, ribavirin, amantadine, rimantadine, cidofovir;antisense oligonucleotides, immunoglobulins and interferons; and drugsfor HIV infection such as tenofovir, emtricitabine, zidovudine,didanosine, zalcitabine, stavudine, lamivudine, nevirapine, delavirdine,saquinavir, ritonavir, indinavir and nelfinavir.

VII. Pharmaceutical Compositions

In in vivo use, generally, whether for immunoimaging, for immunotherapyor by other uses, the conjugate is introduced into a subject. Thecomposition can comprise a single isomer, or one or morepartially-loaded isomers, of the conjugate. For example, if the proteinis an antibody, the composition can comprise a single E2, E4 or E6isomer, a mixture of selected E2, E4 or E6 isomers, all E2, E4 or E6isomers alone, or a mixture of E2, E4 and E6 isomers. In someembodiments, a composition containing a certain isomer(s) can besubstantially free of other isomers. In this context, “substantiallyfree” means the composition contains less than about 20%, less thanabout 10%, less than about 5% less than about 2% or less than about 1%of the other isomers.

The compositions can be in any form that allows for the composition tobe administered to a patient. For example, the composition can be in theform of a solid, liquid or gas (aerosol). Typical routes ofadministration include, without limitation, oral, topical, parenteral,sublingual, rectal, vaginal, ocular, intra-tumor, and intranasal.Parenteral administration includes subcutaneous injections, intravenous,intramuscular, intrasternal injection or infusion techniques. In oneaspect, the compositions are administered parenterally. In yet anotheraspect, the conjugate or compositions are administered intravenously.

The conjugate can be introduced by injection. Typically, the conjugateis administered intravascularly (intravenously or intraarterially) orintrathetically, often by infusion. In addition, in appropriate casesthe conjugate may be introduced subcutaneously, submucosally,intramuscularly, intracranially, or by other accepted routes of drugadministration.

In other embodiments, the composition includes an effective amount of aconjugate and a pharmaceutically acceptable carrier or vehicle. Suchcompositions are suitable for veterinary or human administration.

Pharmaceutical compositions can be formulated so as to allow a conjugateto be bioavailable upon administration of the composition to a patient.Compositions can take the form of one or more dosage units, where forexample, a tablet can be a single dosage unit, and a container of aconjugate in injectable form can hold a plurality of dosage units.

Materials used in preparing the pharmaceutical compositions can benon-toxic in the amounts used. It will be evident to those of ordinaryskill in the art that the optimal dosage of the active ingredient(s) inthe pharmaceutical composition will depend on a variety of factors.Relevant factors include, without limitation, the type of animal (e.g.,human), the particular form of the conjugate, the manner ofadministration, and the composition employed.

The pharmaceutically acceptable carrier or vehicle can be particulate,so that the compositions are, for example, in tablet or powder form. Thecarrier(s) can be liquid, with the compositions being, for example, anoral syrup or injectable liquid. In addition, the carrier(s) can begaseous or particulate, so as to provide an aerosol composition usefulin, e.g., inhalatory administration.

When intended for oral administration, the composition is preferably insolid or liquid form, where semi-solid, semi-liquid, suspension and gelforms are included within the forms considered herein as either solid orliquid.

As a solid composition for oral administration, the composition can beformulated into a powder, granule, compressed tablet, pill, capsule,chewing gum, wafer or the like. Such a solid composition typicallycontains one or more inert diluents. In addition, one or more of thefollowing can be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, or gelatin; excipients such asstarch, lactose or dextrins; disintegrating agents such as alginic acid,sodium alginate, Primogel, corn starch and the like; lubricants such asmagnesium stearate or Sterotex; glidants such as colloidal silicondioxide; sweetening agents such as sucrose or saccharin, a flavoringagent such as peppermint, methyl salicylate or orange flavoring, and acoloring agent.

When the composition is in the form of a capsule, e.g., a gelatincapsule, it can contain, in addition to materials of the above type, aliquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

The composition can be in the form of a liquid, e.g., an elixir, syrup,solution, emulsion or suspension. The liquid can be useful for oraladministration or for delivery by injection. When intended for oraladministration, a composition can comprise one or more of a sweeteningagent, preservatives, dye/colorant and flavor enhancer. In a compositionfor administration by injection, one or more of a surfactant,preservative, wetting agent, dispersing agent, suspending agent, buffer,stabilizer and isotonic agent can also be included.

The liquid compositions, whether they are solutions, suspensions orother like form, can also include one or more of the following: sterilediluents such as water for injection, saline solution, preferablyphysiological saline, Ringer's solution, isotonic sodium chloride, fixedoils such as synthetic mono or digylcerides which can serve as thesolvent or suspending medium, polyethylene glycols, glycerin,cyclodextrin, propylene glycol or other solvents; antibacterial agentssuch as benzyl alcohol or methyl paraben; antioxidants such as ascorbicacid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates orphosphates and agents for the adjustment of tonicity such as sodiumchloride or dextrose. A parenteral composition can be enclosed inampoule, a disposable syringe or a multiple-dose vial made of glass,plastic or other material. Physiological saline is an exemplaryadjuvant. An injectable composition is preferably sterile.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

The amount of the conjugate that is effective in the treatment of aparticular disorder or condition will depend on the nature of thedisorder or condition, and can be determined by standard clinicaltechniques. The dosage ranges for the administration of the disclosedprotein-drug conjugates are those large enough to produce the desiredeffect in which the symptoms of the condition or disorder areameliorated. The dosage should not be so large as to cause adverse sideeffects, such as unwanted cross-reactions, anaphylactic reactions, andthe like. In addition, in vitro or in vivo assays can optionally beemployed to help identify optimal dosage ranges.

The precise dose to be employed in the compositions will also depend onthe age, condition, sex and extent of the disease in the patient, routeof administration, and the seriousness of the disease or disorder, andshould be decided according to the judgment of the practitioner and eachpatient's circumstances.

The compositions comprise an effective amount of a conjugate such that asuitable dosage will be obtained. Typically, this amount is at leastabout 0.01% of a conjugate by weight of the composition. When intendedfor oral administration, this amount can be varied to range from about0.1% to about 80% by weight of the composition. In one aspect, oralcompositions can comprise from about 4% to about 50% of the conjugate byweight of the composition. In yet another aspect, present compositionsare prepared so that a parenteral dosage unit contains from about 0.01%to about 2% by weight of the conjugate.

For intravenous administration, the composition can comprise from about0.01 to about 100 mg of a conjugate per kg of the animal's body weight.In one aspect, the composition can include from about 1 to about 100 mgof a conjugate per kg of the animal's body weight. In another aspect,the amount administered will be in the range from about 0.1 to about 25mg/kg of body weight of the conjugate.

Generally, the dosage of an conjugate administered to a patient istypically about 0.01 mg/kg to about 2000 mg/kg of the animal's bodyweight. In one aspect, the dosage administered to a patient is betweenabout 0.01 mg/kg to about 10 mg/kg of the animal's body weight. Inanother aspect, the dosage administered to a patient is between about0.1 mg/kg and about 250 mg/kg of the animal's body weight. In yetanother aspect, the dosage administered to a patient is between about0.1 mg/kg and about 20 mg/kg of the animal's body weight. In yet anotheraspect the dosage administered is between about 0.1 mg/kg to about 10mg/kg of the animal's body weight. In yet another aspect, the dosageadministered is between about 1 mg/kg to about 10 mg/kg of the animal'sbody weight.

The conjugates can be administered by any convenient route, for exampleby infusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.). Administration can be systemic or local. Various delivery systemsare known, e.g., encapsulation in liposomes, microparticles,microcapsules, capsules, etc., and can be used to administer a conjugateor composition. In certain embodiments, more than one conjugate orcomposition is administered to a patient.

In specific embodiments, it can be desirable to administer one or moreconjugates or compositions locally to the area in need of treatment.This can be achieved, for example, and not by way of limitation, bylocal infusion during surgery; topical application, e.g., in conjunctionwith a wound dressing after surgery; by injection; by means of acatheter; by means of a suppository; or by means of an implant, theimplant being of a porous, non-porous, or gelatinous material, includingmembranes, such as sialastic membranes, or fibers. In one embodiment,administration can be by direct injection at the site (or former site)of a cancer, tumor or neoplastic or pre-neoplastic tissue. In anotherembodiment, administration can be by direct injection at the site (orformer site) of a manifestation of an autoimmune disease.

In certain embodiments, it can be desirable to introduce one or moreconjugates or compositions into the central nervous system by anysuitable route, including intraventricular and intrathecal injection.Intraventricular injection can be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir.

Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent, or viaperfusion in a fluorocarbon or synthetic pulmonary surfactant.

In yet another embodiment, the conjugates can be delivered in acontrolled release system, such as but not limited to, a pump or variouspolymeric materials can be used. In yet another embodiment, acontrolled-release system can be placed in proximity of the target ofthe conjugates, e.g., the brain, thus requiring only a fraction of thesystemic dose (see, e.g., Goodson, in Medical Applications of ControlledRelease, supra, vol. 2, pp. 115-138 (1984)). Other controlled-releasesystems discussed in the review by Langer (Science 249:1527-1533 (1990))can be used.

In some embodiments, a protein conjugate can be combined with a carrierto form a compostion. The term “carrier” refers to a diluent, adjuvantor excipient, with which a conjugate is administered. Suchpharmaceutical carriers can be liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.The carriers can be saline, gum acacia, gelatin, starch paste, talc,keratin, colloidal silica, urea, and the like. In addition, auxiliary,stabilizing, thickening, lubricating and coloring agents can be used. Inone embodiment, when administered to a patient, the conjugate orcompositions and pharmaceutically acceptable carriers are sterile. Wateris an exemplary carrier when the conjugate are administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical carriers also includeexcipients such as starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The compositions, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents.

The compositions can take the form of solutions, suspensions, emulsion,tablets, pills, pellets, capsules, capsules containing liquids, powders,sustained-release formulations, suppositories, emulsions, aerosols,sprays, suspensions, or any other form suitable for use. Other examplesof suitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin.

In an exemplary embodiment, the conjugate is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to animals, particularly human beings.Typically, the carriers or vehicles for intravenous administration aresterile isotonic aqueous buffer solutions. Where necessary, thecompositions can also include a solubilizing agent. Compositions forintravenous administration can optionally comprise a local anestheticsuch as lignocaine to ease pain at the site of the injection. Generally,the ingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where a conjugate isto be administered by infusion, it can be dispensed, for example, withan infusion bottle containing sterile pharmaceutical grade water orsaline. Where the conjugate is administered by injection, an ampoule ofsterile water for injection or saline can be provided so that theingredients can be mixed prior to administration.

Compositions for oral delivery can be in the font), of tablets,lozenges, aqueous or oily suspensions, granules, powders, emulsions,capsules, syrups, or elixirs, for example. Orally administeredcompositions can contain one or more optionally agents, for example,sweetening agents such as fructose, aspartame or saccharin; flavoringagents such as peppermint, oil of wintergreen, or cherry; coloringagents; and preserving agents, to provide a pharmaceutically palatablepreparation. Moreover, where in tablet or pill form, the compositionscan be coated to delay disintegration and absorption in thegastrointestinal tract thereby providing a sustained action over anextended period of time. Selectively permeable membranes surrounding anosmotically active driving compound are also suitable for orallyadministered compounds. In these later platforms, fluid from theenvironment surrounding the capsule is imbibed by the driving compound,which swells to displace the agent or agent composition through anaperture. These delivery platforms can provide an essentially zero orderdelivery profile as opposed to the spiked profiles of immediate releaseformulations. A time-delay material such as glycerol monostearate orglycerol stearate can also be used.

The compositions can be intended for topical administration, in whichcase the carrier may be in the form of a solution, emulsion, ointment orgel base. If intended for transdermal administration, the compositioncan be in the form of a transdermal patch or an iontophoresis device.Topical formulations can comprise a concentration of a conjugate of fromabout 0.05% to about 50% w/v (weight per unit volume of composition), inanother aspect, from 0.1% to 10% w/v.

The composition can be intended for rectal administration, in the form,e.g., of a suppository which will melt in the rectum and release theconjugate.

The composition can include various materials that modify the physicalform of a solid or liquid dosage emit. For example, the composition caninclude materials that form a coating shell around the activeingredients. The materials that form the coating shell are typicallyinert, and can be selected from, for example, sugar, shellac, and otherenteric coating agents. Alternatively, the active ingredients can beencased in a gelatin capsule.

The compositions can consist of gaseous dosage units, e.g., it can be inthe form of an aerosol. The term aerosol is used to denote a variety ofsystems ranging from those of colloidal nature to systems consisting ofpressurized packages. Delivery can be by a liquefied or compressed gasor by a suitable pump system that dispenses the active ingredients.

Whether in solid, liquid or gaseous form, the present compositions caninclude a pharmacological agent used in the treatment of cancer, anautoimmune disease or an infectious disease.

VIII. Pharmokinetics

In vivo characterization of the toxicity and efficacy of purified 4 drugconjugates of cAC10-vcMMAE in mice has been performed and are discussedin more detail in the Examples (see e.g., Examples 8 and 9). Briefly,these studies have shown that mixtures with an average of 4 drugs perantibody are equally efficacious as those conjugates with 8 drugs(single dose of 1 mg/Kg for both), while being less toxic (MTD of 100mg/Kg for 4 drugs per antibody versus 50 mg/Kg for 8 drugs perantibody). The purified material with 4 drugs per antibody from the DTTmethod is similar in efficacy and toxicity, while purified material with4 drugs per antibody from the DTNB method has a slightly higher MTD of120 mg/Kg while being efficacious at half the dose of the otherconjugates (0.5 mg/Kg).

Loading cAC10 with two, four, or eight drugs per antibody had no effecton the binding to the target antigen CD30. The in vitro potency of thecAC10-ADCs was directly dependent on drug loading, and thus the totalMMAE exposure.

cAC10-E4 demonstrated comparable anti-tumor activity to cAC10-E8 in aKarpas-299 xenograft model at the same dose of antibody, half the MMAEdose. Based on the in vitro finding that potency was directly related todrug loading, the equivalent in vivo anti-tumor activity of cAC10-E4 andcAC10-E8 was unanticipated. Investigation of the pharmacokinetics of theADCs revealed that clearance was directly related to the drug loading ofthe ADCs and exposure (Area Under the Curve—AUC) was inversely relatedto drug loading. The AUC of cAC10-E4 was 3-fold higher than cAC10-E8.The larger AUC of cAC10-E4 compared to cAC10-E8 was apparentlysufficient to compensate for the reduced potency, leading to equivalentefficacy. Attempts to improve efficacy by decelerating the plasmaelimination half-life to augment AUCs have been accomplished by methodsincluding the construction of albumin fusion proteins for interferon-αand liposomal delivery of the anti-cancer drug Lurtotecan. Unlike theseexamples where the objective was to lengthen the plasma half-life, theenhanced exposure of cAC10-E4 was a valuable consequence of reducingMMAE loading.

As disclosed in Example 8, dosing cAC10-E2 with 1.0 mg/kg/dose q4dx4yielded ten out of ten cures. While cAC10-E2 did not demonstrateequivalent anti-tumor activity compared to cAC10-E4 at the same mAbdose, the dose of cAC10-E2 to achieve equivalent anti-tumor activitycompared to cAC10-E4 is probably less than two-fold, based on the invivo efficacy experiments. Similar to cAC10-E4, the improved exposure ofcAC10-E2 may play a significant part in compromising the lower in vitropotency.

To maximize the therapeutic potential of cAC10-Val-Cit-MMAE ADCs, a hightherapeutic index is needed. Reducing the amount of MMAE molecules permAb from eight to four enhanced the therapeutic index from 100 to 200.Given steep dose-response curves of chemotherapeutic reagents a two-folddifference in therapeutic index may be significant in terms of theoverall clinical implications with regards to toxicities.

By reducing the quantity of MMAE from eight to four molecules per mAb,there was a decrease of in vitro activity, yet a demonstrated equivalentanti-tumor activity in vivo. While a further reduction in drug loadingto two MMAE molecules per antibody further reduced the in vitroactivity, cAC10-E2 had equivalent or better efficacy than cAC10-E4 andcAC10-E8 at double the dose in a multi-dose setting. The therapeuticwindow was increased two-fold by reducing drug loading from eight MMAEmolecules to four, and at the very least maintained with a furtherreduction to two drugs per antibody. There is considerable value inoptimizing drug substitution of ADCs.

EXAMPLES Example 1 of Method 1

cAC10 was partially reduced with limited concentration of DTT asfollows: cAC10 (8 mg/mL or 53.8 μM) was treated with 3.5 molarequivalents of DTT (188.4 μM; Sigma) in 0.05 M sodium borate pH 8, 0.05M NaCl, and 1 mM diethylene-triaminepentaacetic acid (DTPA; Aldrich) for1 h at 37° C. The reduced antibody was then purified by desalting on aPD-10 column (Amersham Biosciences). The PD-10 column was equilibratedwith 25 mL of phosphate buffered saline (PBS) pH 7.4 (GIBCO) with 1 mMDTPA (PBSD), 1 mL of the above solution applied to the column, thecolumn washed with 1.8 mL of PBSD, and the column eluted with 1.4 mL ofPBSD. The protein concentration was quantitated using an absorbancevalue of 1.58 at 280 nm for a 1.0-mg/mL solution, and the molarconcentration determined using a molecular weight of 150,000 g/mol. Theconcentration of antibody-cysteine thiols produced was determined bytitrating with 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB; Pierce),typically resulting in slightly higher than 4 antibody-cysteine thiolsper antibody using this method.

The drug vcMMAE was then conjugated to reduced cAC10 as follows: reducedcAC10 (typically 30 μM antibody and 120 μM antibody-cysteine thiolsfinal concentration) was first cooled to 0° C. vcMMAE was dissolved incold acetonitrile and rapidly mixed with the antibody solution. Thefinal acetonitrile concentration was 20%, while the final drugconcentration was 135 to 150 μM (4.5 to 5 molar equivalents, which is aslight excess over the antibody-cysteine thiols). This solution wasallowed to incubate for 30 min at 0° C., the excess vcMMAE quenched withcysteine (1 mM final concentration), and the conjugate purified using aPD-10 column as described above.

Example 2 of Method 1

cAC10 with 4 vcMMAE per antibody (E4 mix) was prepared with limitingamounts of DTT as follows: cAC10 was treated with 3.25 molar equivalentsof DTT in 0.025 M sodium borate pH 8, 0.025 M NaCl, 1 mM DTPA for 2 h at37° C. This mixture was diluted 5 fold with water and applied to ahydroxyapatite column (Macroprep ceramic type I 40 μm, BioRad, Hercules,Calif.) at a flow rate of 10 mL/min. The column size was 1 mL per 10 mgof cAC10. The column was previously equilibrated with 5 column volumesof 0.5 M sodium phosphate pH 7, 10 mM NaCl and 5 column volumes of 10 mMsodium phosphate pH 7, 10 mM NaCl. Following application, the column waswashed with 5 column volumes of 10 mM sodium phosphate pH 7, 10 mM NaCland then eluted with 100 mM sodium phosphate pH 7, 10 mM NaCl. DTPA wasadded to 1 mM following elution. The protein concentration wasquantitated using an absorbance value of 1.58 at 280 nm for a 1.0-mg/mLsolution, and the molar concentration determined using a molecularweight of 148,449 g/mol. The concentration of antibody-cysteine thiolsproduced was determined by titrating with DTNB, typically resulting in4.0 to 4.5 thiols per antibody.

Reduced cAC10 was alkylated with a slight excess of vcMMAE overantibody-cysteine thiols (1.1 molar equivalents). To keep the vcMMAEsoluble, 10% DMSO was present in the final reaction mixture.Alternatively, the vcMMAE could be kept in a solution comprising 5% byvolume of an alcohol, such as ethanol and isopropyl alcohol. Thealkylation reaction was performed at 0° C. for 30 min. Cysteine (1 mMfinal) was used to quench any unreacted vcMMAE. cAC10-vcMMAE waspurified by hydroxyapatite chromatography as described above. Followingelution, the buffer was changed to phosphate buffered saline(Invitrogen, Carlsbad, Calif.) using Amicon (Millipore, Bedford, Mass.)Ultrafree 30K cutoff spin concentration devices. The proteinconcentration was quantitated using an absorbance value of 1.62 at 280nm for a 1.0-mg/mL solution.

Example 3 of Method 2a

cAC10 was fully reduced by adding a large excess of DTT. The finalreaction concentrations were 8 mg/mL cAC10, 0.05 M sodium borate pH 8,0.05 M NaCl, 10 mM DTT, and 1 mM DTPA. This solution was incubated at37° C. for 30 min and the antibody purified by desalting on a PD-10column as described above. Slightly more than 8 antibody-cysteine thiolsas determined by DTNB titration were produced using these conditions.

Partial reoxidation was achieved using DTNB as an oxidizing agent.Reduced cAC10 (typically 30 μM) was cooled to 0° C. and then treatedwith 1.5 to 2.5 molar equivalents of DTNB (45 to 75 μM finalconcentration; the highest yields of E4 were obtained using 2.0equivalents). The solution was rapidly mixed by inversion and allowed toincubate at 0° C. for 10-20 min. The extent of reaction can be observedsince the released TNB⁻ is yellow. Typically, the reaction appeared tobe complete within a few seconds. Cysteine was added (1 mM finalconcentration) to ensure that all TNB was present as TNB⁻ rather than inmixed disulfides with antibody cysteines. The antibody was then purifiedon a PD-10 column or a hydroxylapatite column as described above.Typically 4 antibody-cysteine thiols were observed by DTNB titrationfollowing this partial reoxidation procedure. The vcMMAE drug wasfinally conjugated to these antibody-cysteine thiols and purified byPD-10 as described above for method 1.

Example 4 of Method 2b

Fully reduced cAC10 was prepared as described above for method 2a. Fullyreduced cAC10 (typically 30 μM) was cooled to 0° C. and then treatedwith 1.5 to 2.5 equivalents of DTNB (45 to 75 μM final concentration).The solution was rapidly mixed by inversion and allowed to incubate at0° C. for 10 min. Without further purification, the partially reoxidizedcAC10 was then rapidly mixed with 5 equivalents vcMMAE dissolved in coldacetonitrile. As with method 1, the final concentration of acetonitrilewas 20%. In the conjugation reaction, the cAC10 final concentration was24 μM (96 μM antibody-cysteine thiols or 4 per antibody) and the finalvcMMAE concentration was 120 μM (5 molar equivalents). This solution wasincubated for 30 min at 0° C. before quenching with cysteine andpurifying by PD-10 as described above.

Preparative purification of E4 mix by hydroxyapatite. The buffer ofcAC10 (25 mM sodium citrate pH 6.5, 250 mM NaCl, and 0.02% Tween-80) waschanged to PBS using several 15 mL Amicon Ultrafree 30K cutoff spinconcentration devices. 1.09 g of cAC10 in PBS was fully reduced with DTTin a final volume of 89 mL as follows: cAC10 (82.3 μM) was treated with10 mM DTT in 0.025 M sodium borate pH 8, 0.025 M NaCl for 1 h at 37° C.This mixture was diluted to 250 mL with water and applied to a 70 mLhydroxyapatite column (Macroprep ceramic type I 40 μm, BioRad) at a flowrate of 10 mL/min. The column was previously equilibrated with 5 columnvolumes of 0.5 M sodium phosphate pH 7, 10 mM NaCl and 5 column volumesof 10 mM sodium phosphate pH 7, 10 mM NaCl. Following application, thecolumn was washed with 5 column volumes of 10 mM sodium phosphate pH 7,10 mM NaCl and then eluted with 100 mM sodium phosphate pH 7, 10 mMNaCl.

Fully reduced cAC10 was reoxidized with DTNB as follows: eluted materialfrom above (6.02 mg/mL or 40.2 μM, 1.02 g in 170 mL) was cooled to 0° C.and then treated with 2.0 equivalents of DTNB (10 mM stock) for 20 min.Without further purification, reoxidized cAC10 was conjugated to vcMMAE.Cold cAC10 (31.9 μM final) was treated with 5 equivalents of vcMMAE(159.5 μM final) dissolved in DMSO (20% final) in a final volume of 214mL. After 40 min at 0° C., 1.07 mL of 100 mM cysteine was added toquench any unreacted vcMMAE and the mixture was diluted to 750 mL withwater. The conjugate was purified on a hydroxyapatite column asdescribed above for the DTT reduction. The recovered cAC10-vcMMAE E4 mix(0.99 g, or 91% overall yield based on cAC10) was concentrated and thebuffer changed to PBS using several 15 mL Amicon Ultrafree 30K cutoffspin concentration devices.

Preparative purification of pure E4 by HIC was performed on a 45 mLToyopearl phenyl 650M HIC column at a flow rate of 10 mL/min at ambienttemperature. Solvent A was 2 M NaCl and 50 mM sodium phosphate pH 7.Solvent B was 80% v/v 50 mM sodium phosphate pH 7 and 20% v/vacetonitrile. The column was previously equilibrated with 5 columnvolumes of solvent A. Up to 400 mg of cAC10-vcMMAE E4 mix purified byhydroxyapatite (above) was mixed with 1 volume of 4 M NaCl and 50 mMsodium phosphate pH 7 and applied to the column. E0 was not retained bythe column. The different drug loaded species were eluted by sequentialstep gradients: E2 was eluted with 35% solvent B, E4 was eluted with 70%solvent B, E6 was eluted with 95% solvent B, and E8 was eluted with 100%solvent B. Purified E4 was concentrated and the buffer changed to PBSusing several 15 mL Amicon Ultrafree 30K cutoff spin concentrationdevices, yielding 235 mg of pure E4 from two 400 mg purifications.Purity analysis by analytical HIC (below) showed E4 purity greater than90%.

Example 5 of Method 1

TCEP limited reduction followed by alkylation without intermediatepurification was accomplished by treating cAC10 with 2.75 molarequivalents of TCEP in 0.025 M sodium borate pH 8, 0.025 M NaCl, 1 mMDTPA for 2 h at 37° C. See also FIG. 9 The mixture was then cooled to 0°C., and partially reduced cAC10 was alkylated with vcMMAE as describedabove. cAC10-vcMMAE was desalted using PD-10 columns (AmershamBiosciences, Piscataway, N.J.) equilibrated with phosphate bufferedsaline. (Partial reduction can also be performed with an intermediatepurification step of the partially reduced antibody, as shown in FIG.8.)

The samples used to determine the kinetics of isomer distribution wereprepared as follows: cAC10 was reduced with 3.0 equivalents of DTT in 50mM sodium phosphate pH 7.5 and 5 mM EDTA at 37° C. At the indicated timepoints, samples were removed, quenched with an equal volume of 200 mMsodium citrate pH 5, and purified using PD-10 columns equilibrated withphosphate buffered saline containing 5 mM EDTA. Reduced cAC10 wastreated with vcMMAE as previously described and purified using PD-10columns equilibrated with phosphate buffered saline.

Purification of E2, E4, and E6 pure by HIC was performed on a Toyopearlphenyl 650M HIC column (Tosoh Biosciences, Montgomeryville, Pa.) at aflow rate of 10 mL/min at ambient temperature. The column size was 1 mLper 7.5 mg of cAC10-vcMMAE. Solvent A was 2.0 M NaCl and 50 mM sodiumphosphate pH 7. Solvent B was 80% v/v 50 mM sodium phosphate pH 7 and20% v/v acetonitrile. The column was previously equilibrated with 5column volumes of solvent A. cAC10-vcMMAE was mixed with 0.67 volume of5 M NaCl (2.0 M final) and applied to the column. E0 was not retained bythe column. The different drug loaded species were eluted by sequentialstep gradients: E2 was eluted with 35% solvent B, E4 was eluted with 70%solvent B, E6 was eluted with 95% solvent B, and E8 was eluted with 100%solvent B. Purified E4 was concentrated and the buffer changed tophosphate buffered saline using Ainicon Ultrafree 30K cutoff spinconcentration devices.

Example 6 of Analytical Methods

Drug loading was determined by measuring the ratio of the absorbance at250 and 280 nm (A250/280). The number of vcMMAE per cAC10 has beenempirically determined to be (A250/280−0.36)/0.0686.

The conjugates were analyzed for percent E4 purity by hydrophobicinteraction chromatography (HIC) using a Tosoh Biosceince Ether-5PWcolumn (part 08641) at a flow rate of 1 mL/min and a column temperatureof 30° C. Solvent A was 50 mM sodium phosphate pH 7 and 2 M NaCl.Solvent B was 80% 50 mM sodium phosphate pH 7, 10% 2-propanol, and 10%acetonitrile. Isocratic 0% B for 15 min, a 50-min linear gradient from 0to 100% B, a 0.1-min linear gradient from 100 to 0% B, and isocratic 0%B for 14.9 min. Injections (typically 90-100 μL) were 1 volume ofpurified vcMMAE-cAC10 conjugate (concentration of at least 3 mg/mL) and1 volume of 50 mM sodium phosphate pH 7 and 4 M NaCl.

The ADC's, including pure E4 from HIC chromatography, were analyzedunder denaturing and non-reducing conditions using an AgilentBioanalyzer. A protein 200 chip was used under denaturing butnonreducing conditions as described by the manufacturer. Briefly, 4 μLof 1 mg/mL cAC10-vcMMAE was mixed with 2 μL of nonreducing loadingbuffer and heated to 100° C. for 5 min. Water (84 μL) was added and 6 μLof this mixture was loaded into each well of the chip.

Pure E4 was finally analyzed on a PLRP-S column (Polymer Laboratories).The flow rate was 1 mL/min and the column temperature was 65° C. SolventA was 0.05% trifluoroacetic acid in water and solvent B was 0.04%trifluroacetic acid in acetonitrile. Isocratic 25% B for 3 min, a 15-minlinear gradient to 50% B, a 2-min linear gradient to 95% B, a 1-minlinear gradient to 25% B, and isocratic 25% B for 2 min. Injections were10 μL of cAC10-vcMMAE previously reduced with 20 mM DTT at 37° C. for 20min to cleave the interchain disulfides.

The ADC's also were analyzed under denaturing and reducing conditions ona PLRP-S column (Polymer Laboratories) (2.1×150 mm, 8 μ, 1000 Å). Theflow rate was 1 mL/min and the column temperature was 80° C. Solvent Awas 0.05% trifluoroacetic acid in water and solvent B was 0.04%trifluroacetic acid in acetonitrile. Isocratic 25% B for 3 min, a 25-minlinear gradient to 50% B, a 2-min linear gradient to 95% B, a 1-minlinear gradient to 25% B, and isocratic 25% B for 2 min. Injections were10-20 μL of I mg/ml cAC10-vcMMAE previously reduced with 20 mM DTT at37° C. for 15 min to cleave the remaining interchain disulfides. Themole fraction of each chain was determined using the following molarextinction coefficients: light chain with 0 vcMMAE: 30,160 M⁻¹ cm⁻;light chain with 1 vcMMAE: 31,660 M⁻¹ cm⁻¹; heavy chain with 0 vcMMAE:86,915 M⁻¹ cm⁻¹; heavy chain with 1 vcMMAE: 88,415 M⁻¹ cm⁻¹; heavy chainwith 2 vcMMAE: 89,915 M⁻¹ cm⁻¹; heavy chain with 3 vcMMAE: 91,415 M⁻¹cm⁻¹.

The isomeric distribution for E2 and E6 was determined using solelyPLRP-S HPLC data. For E2 isomer A (for these analyses, “isomer A” refersto both isomers 2A and 2B of FIG. 7), the mole fraction of light chainwith 0 vcMMAE (L0) is equal to the mole fraction of heavy chain with 1vcMMAE (H1), while for E2 isomer C, the mole fraction of light chainwith 0 and 1 vcMMAE and the mole fraction of heavy chain with 0 and 1vcMMAE are all equal. Since only light chain with 1 vcMMAE (L1) andheavy chain with 0 vcMMAE (HO) contribute to the percentage of isomer C,the percent isomer C can be expressed as follows:

% C=2L1+2H0   (1)

The percent of isomer A is assumed to be 100-% C. Small amounts (lessthan 3% total) of heavy chain with 2 or 3 vcMMAE are often observed inthe PLRP-S HPLC data. These are probably due to contaminating E4 or E6in the E2 sample. For the purposes of calculating the percent of E2isomers A and C, the sum of the mole percent of L0, L1, H1, and H2 wasset to 100%.

Similarly, for E6 isomer A (for these analyses, “isomer A” refers toboth isomers 6A and 6B of FIG. 7), the mole fraction of H2 is equal tothe mole fraction of L1, while for E6 isomer C, the mole fractions ofL0, L1, H2, and H3 are equal. Since only L0 and H3 contribute to thepercentage of isomer C, the percent isomer C can be expressed asfollows:

% C=2L0+2H3   (2)

The percent of isomer A is assumed to be 100-% C. As with E2, the sum ofthe mole percent of L0, L1, H2, and H3 was set to 100%.

The percentages of the E4 isomers cannot be obtained solely from thePLRP-S HPLC data because there is not a unique solution. At least oneisomer needs to be fixed before PLRP data can be used to solve for theother two isomers. The mole percent of HHL, HH, and HL were determinedfrom Bioanalyzer data using the following molecular weights: 124,720.8(HHL), 100,992.6 (HH), and 74,224.5 (HL) g/mol. The Bioanalyzer uses thefluorescence of bound dye for instrument readout, and it is assumed thatHHL, HH, and HL bind the dye equally per unit molecule weight, althoughit is unlikely that this assumption is true. To minimize the error thatwould result from this assumption, only isomer E4A was calculated fromBioanaylzer data using the HHL, HH, and HL peak areas as follows (the HLpeak area is divided by 2 since each antibody would produce 2 HL if theheavy-heavy chain disulfides were cleaved):

$\begin{matrix}{{\% \mspace{14mu} A} = \frac{\frac{H\; H\; L}{124720.8}}{\frac{H\; H\; L}{124720.8} + \frac{H\; H}{100992.6} + \frac{H\; L}{2*74224.5}}} & (3)\end{matrix}$

PLRP-S HPLC data was then used to solve for the remaining contributionof E4 isomers E and F using the following formulas:

% E=H1+L1−0.5% A   (4)

% F=H2+L0−0.5% A   (5)

E4 isomer A contributes equally to the populations of L0, L1, H1, andH2, and the H1 and L1 contributions of isomer A (half of its totalcontribution) must be subtracted from the total observed amount of H1and L1 to give the remaining amount of H1 and L1 that must be due to thepresence of isomer E. A similar subtraction for the contribution of H2and L0 for isomer A will yield the amount present due to isomer F. Aswith E2 and E6, the sum of the mole percent of L0, L1, H1, and H2 wasset to 100%.

Example 7 of Strategies for Partial Loading of Protein

Two different strategies were used to prepare partially drug loadedADCs. First, partial reduction of cAC10 with limiting amounts of DTT orTCEP yields fewer than 8 antibody cysteines. About 3.25 and 2.75equivalents of DTT and TCEP, respectively, will cleave 2 interchaindisulfide bonds to yield an average of 4 cAC10 cysteines per antibody (amixture of 0, 2, 4, 6, and 8 antibody-cysteines). The amount of reducingagent can be empirically determined: cBR96 requires only 2.1 equivalentsof DTT or TCEP to yield 4 antibody cysteines, while murine IgG1antibodies can often by extremely resistant to reduction (data notshown). An advantage of using TCEP rather than DTT is that phosphinesreact poorly with maleimides, and any remaining reducing agent does nothave to be removed before adding vcMMAE. Excess DTT readily reacts withvcMMAE and would compete with antibody-cysteines for the drug. Followingantibody reduction, treatment of antibody cysteines with a slight molarexcess of vcMMAE (1.1 molar equivalents per cysteine) yields cAC10 withan average drug loading of 4 MMAE per antibody (E4 mix).

Alternatively, cAC10 can be fully reduced with 10 mM DTT and thenpartially reoxidized with DTNB. This reoxidiation process is veryefficient, requiring 2.0 equivalents of DTNB to reoxidize 8 antibodycysteines to 4. Treatment of this reoxidized antibody with a thiol suchas cysteine does not liberate any bound thionitrobenzoic acid,suggesting that the reoxidized cysteines are in the form of antibodydisulfides rather than mixed TNB-cysteine disulfides. The analyticalmethods described below also show the presence of antibody disulfides.The remaining antibody cysteines can be conjugated to vcMMAE asdescribed above to yield E4 mix.

To determine the isomeric population of each of the drug loaded species,E2, E4, and E6, are separated and isolated, yielding E2, E4, and E6pure. FIG. 13A shows a hydrophobic interaction (HIC) HPLC trace of E4mix made by DTT partial reduction. All of the even drug loaded speciescan be separated from each other, and small amounts of odd drug loadedspecies can be seen in the trough between the even species. The dragloading of these species can be assigned by inspection of the UV spectraof the peaks. The PABA group in the drug linker has a maximum absorbancenear 248 mu, while the antibody has a minimum absorbance at the samewavelength. Using the drug and antibody extinction coefficients at 248and 280 nm, the number of drugs per antibody can be assigned for thestarting ADC mixture and each of the observed peaks (Hamblett et al.(2004), Clin Cancer Res 10: 7063-70).

Table 1 shows the percentages of the even drug loaded species preparedby DTT partial reduction, TCEP partial reduction, and partial DTNBreoxidation. The DTNB partial reduction method yields a slightly higherpercentage of E4 (38%) than the partial reduction methods (30% for DTTand 33% for TCEP). This comes at the expense of mainly E6 and E8, whichtotal about 34% for DTT partial reduction and 31% for TCEP partialreduction, while only 24% for DTNB partial reoxidation. The odd drugloaded species not shown on the table and account for 7-10% of the totalmaterial.

TABLE 1 Percent composition of E4 mixture.^(a) Production method E0 E2E4 E6 E8 DTT partial 9 ± 2 20 ± 3 30 ± 1 24 ± 3 10 ± 3  reduction TCEPpartial 8 ± 1 20 ± 3 33 ± 2 22 ± 2 9 ± 1 reduction DTNB partial 10 ± 4 18 ± 3 38 ± 2 20 ± 4 4 ± 2 reoxidiation ^(a)HIC-HPLC chromatograms wereintegrated for percent composition. Values are plus or minus standarddeviation for 4 (DTT partial reduction), 3 (TCEP partial reduction), or6 (DTNB partial reoxidation) separate batches. The contributions fromodd species are not shown, causing the total to be less than 100%.

This HIC-HPLC method can be used to isolate a few milligrams of E2, E4,and E6 pure. Alternatively, preparative HIC using step gradients can beused to isolate hundreds of milligrams of E2, E4, and E6 pure, as shownin FIGS. 13B, C, D. The purity of these materials, with respect to theirdrug loading levels, is at least 95%.

These purified materials were subjected to two analytical methods todetermine the distribution of the drugs on the antibody (see Example 6).First, reducing and denaturing HPLC on a PLRP-S column was used todetermine the number of drugs per antibody chain. Pretreatment of theADC with an excess of DTT breaks the remaining interchain disulfides andallows separation of light chain with 0 or 1 drugs (L0 and L1) fromheavy chain with 0, 1, 2, or 3 drugs (H0, H1, H2, and H3) (FIG. 4).Second, non-reducing and denaturing capillary electrophoresis allowsseparation of antibody chains with the remaining interchain disulfidesintact, resulting in 6 potential species: L, H, HL, HH, HHL, and HHLL(FIG. 15).

Quantitation of the species observed by PLRP-S HPLC and capillaryelectrophoresis allows assignment of the isomeric populations. FIGS. 1and 7 illustrate the antibody fragments and the number of associateddrugs for each of the isomers. The isomeric populations of E2 and E6 caneasily be determined by PLRP-S HPLC alone or capillary electrophoresisalone because each isomer yields a unique pattern. For instance, onlyisomer E2C yields L1 and H0 under denaturing and reducing conditions,while E2A only yields L0 and H1, and under denaturing and non-reducingconditions isomer E2A yields HHLL while E2C yields L and HHL. For E4,neither PLRP-S HPLC nor capillary electrophoresis alone is sufficient tocalculate the isomeric populations, so the two methods must be used incombination to determine the composition. Table 2 shows the percentcomposition for each of these isomers. PLRP-S HPLC data was usedexclusively for calculating the isomeric composition of the E2 and E6isomers using Equations 1 and 2 (see Example 6). Capillaryelectrophoresis was used to calculate the amount of E4A using Equation3, and PLRP-S HPLC was used to calculate the amount of E4B and E4C usingEquations 4 and 5 which subtract out the contribution of E4A (seeExample 6).

TABLE 2 Composition of isomeric population of purified E2, E4, and E6.Production method E2A^(a) E2C^(a) E4A^(b) E4E^(c) E4F^(c) E6A^(d)E6C^(d) DTT partial 8 92 10 59 31 2 98 reduction DTNB partial 77 23 17 875 4 96 reoxidation AET pH 5 partial 17 83 13 46 41 2 98 reduction^(a)Determined from PLRP-S HPLC data using Equation 1. ^(b)Determinedfrom Bioanalyzer data using Equation 3. ^(c)Determined from PLRP-S HPLCdata using Equations 4 and 5. ^(d)Determined from PLRP-S HPLC data usingEquation 2.

The data in Table 2 is striking because the production methodsignificantly effects the location of the drugs, suggesting that theantibody disulfides can be selectively reduced. Partial DTT reductionyields 92% isomer E2C, which results from reduction of one of theheavy-light chain disulfides, 59% isomer E4E, which results from thereduction of both heavy-light chain disulfides, and 98% isomer E6C,which results from reduction of both heavy-heavy chain disulfides andone heavy-light chain disulfide. Isomers with one heavy-heavy chaindisulfide reduced are in the extreme minority. On the other hand,partial DTNB reoxidation yields almost the opposite result for E2 and E4isomers, 77% isomer E2A and 75% MA, where one heavy-heavy chaindisulfide is intact, and the same result for E6, 96% E6C. Acidicreduction with AET yields an isomer population that is very similar toDTT partial reduction, and favors cleavage of the heavy-light chaindisulfides.

The kinetics of the isomer distribution for DTT partial reduction isshown in Table 3. cAC10 was reduced with 3.0 equivalents of DTT andsamples were periodically removed and alkylated with vcMMAE. E2, E4, andE6 pure were obtained by HIC-HPLC, and the isomer distribution wasdetermined by PLRP-S HPLC and Bioanaylzer. The isomer compositions areidentical over the course of the experiment, covering 10 to 120 min ofreduction time and a total drug loading of 1.3 to 3.9 drugs perantibody. These results show that the DTT partial reduction isomerpopulations shown in Table 2, prepared by reducing cAC10 for 2 h with alimiting amount of DTT, are representative of the isomeric populationover the entire course of the reduction reaction.

TABLE 3 Kinetics of isomer distribution for DTT partial reduction. Time(min)^(a) Drugs/mAb^(b) E2A^(c) E2C^(c) E4A^(d) E4E^(e) E4F^(e) E6A^(f)E6C^(f) 10 1.3 12 88 9 63 28 N/D N/D 20 2.1 9 91 7 65 29 7 93 35 2.7 991 7 63 31 6 94 55 3.3 9 91 7 63 30 8 92 80 3.6 9 92 7 61 32 6 94 1203.9 11 90 8 61 31 7 93 ^(a)Reduction time. Once reduced, all antibodieswere treated with vcMMAE for identical times. ^(b)Determined byHIC-HPLC. ^(c)Determined from PLRP-S HPLC data using Equation 1.^(d)Determined from Bioanalyzer data using Equation 3. ^(e)Determinedfrom PLRP-S HPLC data using Equations 4 and 5. ^(f)Determined fromPLRP-S HPLC data using Equation 2. N/D, not determined. At this timepoint, very little E6 was produced and this material was not sufficientfor determining the isomer population.

TABLE 4 In vitro binding and cytotoxicity of cAC10-vcMMAE. ADC BindingIC₅₀ (μg/mL)^(a) Karpas 299 IC₅₀ (ng/mL)^(b) E0 (cAC10) 2.70 ± 1.91 N/DE2 mix DTT N/D 11.4 ± 2.4  E2 pure DTT 3.57 ± 2.41 13.8 ± 3.6  E2 mixDTNB N/D 11.7 ± 4.5  E2 pure DTNB 2.02 ± 1.22 13.2 ± 2.7  E4 mix DTT N/D3.4 ± 1.2 E4 pure DTT 7.76 ± 3.93 4.8 ± 0.7 E4 mix DTNB N/D 5.0 ± 0.0 E4pure DTNB 7.69 ± 4.42 4.3 ± 0.9 E8 6.53 ± 3.09 2.7 ± 0.2 ^(a)Binding toKarpas 299, in μg of antibody component/mL, determined from 4-7independent measurement plus or minus the standard deviation. N/D, notdetermined. ^(b)In vitro cytotoxicity, in ng of antibody component/mL,determined from 3 independent measurements plus or minus the standarddeviation. N/D, not determined. cAC10 alone displays poor potencyagainst Karpas 299.

Table 4 lists the results of in vitro binding and cytotoxicityexperiments that were performed for ADCs of several drug loading levels.E2 and E4 mix as well as E2 and E4 pure from DTT partial reduction andDTNB partial reoxidation were tested. The fully loaded conjugate with 8drugs per antibody was the most cytotoxic, with an IC50 value on theCD30 positive Karpas 299 cell line of 2.7 ng/mL (calculated based on theweight of the antibody). The ADCs with 4 drugs per antibody wereslightly less cytotoxic, with IC50 values between 3.4 and 5.0 ng/mL, andthe ADCs with 2 drugs per antibody were the least cytotoxic, with IC50values between 11.4 and 13.8 ng/mL. The chemistry used to produce theADCs did not show any significant differences in the cytotoxicity, norwere there significant differences between the mixtures and theHIC-purified ADCs. The in vitro cytotoxicity appears to depend only onthe total dose of drug. Binding to CD30 positive cells was very similarfor E0, E2, and E4, with E8 being slightly impaired, demonstrating thatconjugation does not interfere with antigen binding. The in vitrocytotoxicities of the ADCs (measuring the antibody component) show theexpected trend: the larger the number of drugs, the lower the IC50value. Within the error of the experiment, the location of the drugsdoes not appear to influence the in vitro cytotoxicity.

Example 8 of Drug Loading Effects on Antitumor Activity of MonoclonalAntibody Drug Conjugate

Cells and reagents. CD30-positive ALCL line Karpas-299 was obtained fromthe Deutsche Sammlung von Mikroorganism and Zellkulturen GmbH(Braunschweig, Germany). L540cy, a derivative of the HD line L540adapted to xenograft growth, was graciously provided by Dr. Harald Stein(Institut fur Pathologie, Univ. Veinikum Benjamin Franklin,Hindenburgdamm 30, 12200 Berlin, Germany). Cell lines were grown inRPMI-1640 media (Life Technologies Inc., Gaithersburg, Md.) supplementedwith 10% fetal bovine serum.

Construction and purification of cAC10-Val-Cit-MMAE ADCs. Briefly, cAC10with 8 drugs per antibody was produced by cAC10 was mixed withdithiothreitol (DTT) at 37° C. for 30 min, and the buffer was exchangedby elution through Sephadex G-25 resin with PBS containing 1 mMdiethylenetriaminepentaacetic acid (DTPA). PBS containing 1 mM DTPA(PBS/D) was added to the reduced mAb (final concentration 2.5 mg/mL). A9.5 molar excess of maleimidocaproyl-Val-Cit-MMAE, referred to asVal-Cit-MMAE, was added to the reduced antibody at 4° C. for 1 h and theconjugation reaction was quenched by adding a 20-fold excess ofcysteine. The reaction mixture was concentrated by centrifugalultrafiltration and buffer-exchanged through Sephadex G25 equilibratedin PBS at 4° C. The conjugate was then filtered through a 0.2 micronfilter under sterile conditions.

The generation of cAC10 ADCs with two and four MMAE molecules perantibody involved a partial reduction followed by reaction withVal-Cit-MMAE. The antibody cAC10 (10 mg/ml) was partially reduced byaddition of DTT to a final DTT:mAb molar ratio of 3.0 followed byincubation at 37° C. for ˜2 h. The reduction reaction was then chilledto ˜10° C. and the reduced cAC10 purified away from excess DTT viadiafiltration. Following diafiltration, the thiol concentration in thepartially-reduced cAC10 was determined by the DTNB (Elhnan's) assay; inthis manner, an average of about 2 disulfide bonds were reduced, thusexposing about 4 reduced Cys:mAb. To conjugate all of the reduced Cys,Val-Cit-cMMAE was added to a final Val-Cit-MMAE:reduced Cys molar ratioof about 1.15. The conjugation reaction was carried out in the presenceof 15% v/v of DMSO and allowed to proceed at about 10° C. for about 30min. Following the conjugation reaction, excess free Cys (2 moles of Cysper mole of Val-Cit-MMAE) was added to quench unreacted Val-Cit-MMAE toproduce the Cys-Val-Cit-MMAE adduct. The Cys quenching reaction wasallowed to proceed at about 10° C. for about 30 min. The Cys-quenchedreaction mixture was purified and buffer-exchanged into PBS bydiafiltration to obtain the partially loaded cAC10-Val-Cit-MMAE.

Preparative HIC fractionation. All chromatographic steps were performedat room temperature. A 1.6×25 cm column (˜50 ml) was packed withToyopearl Phenyl-650M HIC resin (Tosoh Bioscience, Montgomeryville, Pa.)and equilibrated with >5 column volumes of Buffer A (50 mM sodiumphosphate, 2 M NaCl, pH 7.0) at a flow rate of 15 ml/min. To prepare thesample for loading onto the column, 39 ml of partially loadedcAC10-vcMMAE (12.9 mg/ml) was blended with 39 ml of Buffer A′ (50 mMsodium phosphate, 4 M NaCl, pH 7.0). The sample was loaded onto thecolumn at 10 ml/min; all subsequent steps were performed at a flow rateof 10 ml/min. Following sample loading, the column was washed withBuffer A until an A₂₈₀ baseline was achieved. cAC10-E2 was eluted andcollected with a step gradient consisting of 65% Buffer A/35% Buffer B(80% v/v 50 mM sodium phosphate, pH 7.0, 20% v/v acetonitrile). Afterbaseline was again achieved, cAC10-E4 was eluted and collected with astep gradient consisting of 30% Buffer A/70% Buffer B. Both cAC10-E2 andcAC10-E4 peaks were collected to ˜20% of their respective peak heights.The fractions of interest were buffer exchanged into PBS usingUltrafree-15 centrifugal filter devices with a molecular weight cutoffof 30 kDa (Millipore, Billerica, Mass.).

Analysis of conjugates. Analysis of the conjugates was accomplished byHIC-HPLC using an Ether-5PW column (Tosoh Bioscience, Montgomeryville,Pa.). The method consisted of a linear gradient from 100% Buffer A to100% Buffer C (80% v/v 50 mM sodium phosphate, pH 7.0, 10% v/vacetonitrile, 10% v/v isopropanol) in 50 min. The flow rate was set at 1ml/min, the temperature was set at 30° C., and detection was followed atboth 248 and 280 nm. The identity of unmodified cAC10 and cAC10-E8 peakswas confirmed by injection of cAC10 and cAC10-E8 standards. Because theantibody and drug have distinct absorbance maxima (ο_(max)=280 and 248nm, respectively), it was possible to identify peaks corresponding tocAC10 conjugates with 2, 4, and 6 drugs per antibody by overlaying peakspectra.

In vitro characterization of cAC10-Val-Cit-MMAE ADCs. Competitionbinding was performed on the ADCs to determine if the conjugation orpresence of drug affected the antigen binding. To compare saturationbinding of mAb and ADC, 5×10⁵ Karpas-299 cells were combined with serialdilutions of cAC10, cAC10-E2, cAC10-E4, or cAC10-E8 in the presence of 1μg/ml cAC10 labeled with Alexa Fluor 488 (Molecular Probes, Eugene,Oreg.) in staining medium for 30 min on ice and washed twice with icecold staining medium. Labeled cells were examined by a Fusion microplatereader (Perkin-Elmer, Boston, Mass.). Sample data werebackground-corrected and reported as the percent of maximum fluorescenceas calculated by the sample fluorescence divided by the fluorescence ofcells stained with 1 μg/mL cAC10-Alexa Fluor® 488 alone.

The growth inhibitory activities of cAC10 conjugates were determined bymeasuring DNA synthesis. Conjugates were incubated with CD30⁺ Karpas-299or L540cy cells or CD30⁻ WSU-NHL cells. After a 92 h incubation withcAC10 or cAC10 ADCs cells were labeled with [³H]-thymidine, 0.5μCi/well, for 4 h at 37° C. Cells were harvested onto filters using aharvester, mixed with scintillation fluid and the radioactivity wasmeasured with a Topcount scintillation counter (Packard Instruments,Meriden, Conn.). The percent untreated was plotted versus concentrationfor each molecule to determine the IC₅₀ (defined as the mAbconcentration that gave 50% inhibition of DNA synthesis).

Xenograft models of human ALCL. To establish a subcutaneous diseasemodel of ALCL 5×10⁶ Karpas-299 cells were implanted into the right flankof CB-17 SOD mice (Harlan, Indianapolis, Ind.). Therapy with ADCs wasinitiated when the tumor size in each group of 6-10 animals averagedapproximately 50-100 mm³. Treatment consisted of either a singleinjection or multiple i.v. injections using the schedule of oneinjection every 4 days for 4 injections (q4dx4). Tumor volume wascalculated using the formula (length×width²)/2. A tumor that decreasedin size such that it was impalpable was defined as a complete regression(CR). A complete regression that lasted for ≧10 tumor doubling times wasdefined as a cure. Tumor growth inhibition (TGI) was calculated whentumors in the control group reached 750-1000 mm³ in size as follows:

${TGI} = {1 - {\frac{\left( {{Mean}\mspace{14mu} {tumor}\mspace{14mu} {volume}\mspace{14mu} {of}\mspace{14mu} {treated}\mspace{14mu} {group}} \right)}{\left( {{Mean}\mspace{14mu} {tumor}\mspace{14mu} {volume}\mspace{14mu} {of}\mspace{14mu} {control}\mspace{14mu} {group}} \right)} \times 100}}$

Maximum tolerated dose. Groups of three BALB/c mice (Harlan,Indianapolis, Ind.) were injected with 30-60 mg/kg of cAC10-E8, 60-120mg/kg of cAC10-E4, or 200-250 mg/kg of cAC10-vcMMAE2 via the tail veinto determine the single dose maximum tolerated dose (MTD). Mice weremonitored daily for 14 days, and both weight and clinical observationswere recorded. Mice that developed significant signs of distress weresacrificed in accordance with ACUC guidelines. The maximum tolerateddose was defined as the highest dose which did not cause a serious overttoxicities or greater than 20 percent weight loss within two weeks ofinjection in any of the animals.

Pharmacokinetics. The pharmacokinetics of cAC10, cAC10-E2, cAC10-E4, andcAC10-E8 were evaluated in SOD mice. SCID mice (n=3) were administered10 mg/kg of test material (based on the antibody component) by tail veininjection. Blood samples were collected from each mouse via thesaphenous vein at 1 h, 4 h, 1 d, 2 d, 4 d, 7 d, 14 d, 21 d, 28 d, 35 d,42 d, and 49 days post injection. Blood was collected into heparincoated tubes followed by centrifugation (14,000×g, 3 min) to isolateplasma. Plasma concentrations of cAC10 and ADCs were measured by ELISA.

Briefly, the ELISA consisted of the following steps: plate coat, block,sample binding, secondary mAb, TMB, and acid stop. After each step thewells were washed with wash buffer (PBS, 0.05% Tween-20, pH=7.4) threetimes. In the plate coat step anti-cAC10 mAb was coated onto 96-wellplates at 2 μg/mL in carbonate buffer (0.1 M carbonate/bicarbonate,pH=9.6) at 4° C. overnight. Following the plate coat, blocking buffer(PBS, 1% BSA, 0.05% Tween-20) was added and incubated at roomtemperature for 1 h. Next, 100 μL of standard or diluted plasma samplewas added to triplicate wells for 1 h at room temperature. The secondarystep consisted of a mouse anti-human IgG-HRP conjugate (SouthernBiotech, Birmingham, Ala.) incubated for 1 h. Subsequently, 100 μL of3,3′,5,5′-tetramethylbenzidine (Sigma, St. Louis, Mo.) was added to eachwell and upon color development the reaction was stopped with 100 μL of1 N sulfuric acid. Optical density was measured using a VMax KineticMicroplate reader (Molecular Devices, Sunnyvale, Calif.) at 450 nm and ablank at 630 nm. Non-compartmental pharmacokinetic parameters werecalculated with WinNonlin (Pharsight, Mountain View, Calif.).

In vitro characterization. Competition binding experiments wereperformed to evaluate if conjugation of MMAE to cAC10 interfered withthe CD30 binding capability of the ADCs. CD30⁺ Karpas-299 cells wereincubated with 1 pg/mL of fluorescently labeled cAC10 combined withserial dilutions of unlabeled antibody, cAC10-E2, cAC10-E4, or cAC10-E8.As shown in FIG. 10, each of the ADC variants effectively competed withfluorescently labeled cAC10 equivalent to unlabeled cAC10. Thus,conjugation with MMAE did not reduce antigen binding.

The in vitro cytotoxic activities of the ADCs were evaluated by a[³H]-TdR incorporation assay with CD30⁺ Karpas-299 and L540cy cells andCD30⁻ WSU-NHL cells. cAC10-E8 demonstrated significant activity againstthe Karpas-299 cells with an IC₅₀ of 1.0 ng/mL (FIG. 11 a). Decreasingthe amount of drug in half to four MMAE molecules per mAb (cAC10-E4)increased the IC₅₀ to 2.9 ng/mL. Halving the drug loading again furtherincreased the IC₅₀ to 6.2 ng/mL with cAC10-E2. Against the HD lineL540cy the ADCs had a similar trend with 10₅₀ values of 1.4, 4.5, and9.2 ng/mL for cAC10-E8, cAC10-E4, and cAC10-E2, respectively (FIG. 11b). Selectivity of the

ADCs was evaluated with CD30⁻ WSU-NHL cell line which were insensitiveto all cAC10-ADCs with IC₅₀ values >1000 ng/ml (data not shown).

Xenograft models of human ALCL. The effect of drug loading on in vivoanti-tumor activity was evaluated with a Karpas-299 subcutaneousxenograft models. Therapy was administered every fourth day for a totalof 4 injections (q4dx4) starting when tumor volumes reached 50-100 mm³.Using this schedule, it was previously found that cAC10-E8 at 1 mg/kgproduced 100% CRs, at the same dose cAC10-E4 obtained 100% CRs (data notshown). With the goal of comparing the activity of the ADCs, lower doseswere used for cAC10-E4 and cAC10-E8. Cohorts of mice bearingsubcutaneous Karpas-299 xenografts were treated with multiple doses ofcAC10-E2 (0.5 mg/kg/dose or 1 mg/kg/dose), cAC10-E4 (0.25 or 0.5mg/kg/dose), or cAC10-E8 (0.25 or 0.5 mg/kg/dose). Table 5 displays asummary of the efficacy results.

TABLE 5 Table 5: Anti-tumor activity of cAC10-E2, cAC10- E4,cAC10-E4-Mixture, and cAC10-E8 in a subcutaneous Karpas-299 xenograftmodel. Dose Complete Schedule ADC (mg/kg) Regressions Cures TGI q4dx4cAC10-E2 0.5 0/10 0/10 68% 1.0 10/10  10/10  97% cAC10-E4 0.25 1/10 1/1056% 0.50 5/10 3/10 91% cAC10-E8 0.25 0/10 0/10 47% 0.50 6/10 6/10 90% x1cAC10-E2 1.0 4/6  4/6  96% cAC10-E4 1.0 6/6  5/6^(a ) 100% cAC10-E8 1.05/6  5/6  100% x1 cAC10-E4 1.0 9/10 7/10 99.2% cAC10-E4- 1.0 8/10 8/1098.4% Mixture Animals were treated with ADCs once tumor volumes reached50-100 mm³. Doses were given once (x1) or four times every four days(q4dx4). The number of complete regressions, cures and tumor growthinhibition (TGI) are reported. ^(a)one mouse with a cure was found deadon Day 72 with no sign of tumor mass.

While cAC10-E8 had twice the amount of MMAE as cAC10-E4 at the same mAbdose, they were equally effective at both dose levels (FIG. 12A). At the0.5 mg/kg/dose, out of the ten animals treated with cAC10-E4 fiveachieved complete regressions (CRs) and cAC10-E8 induced six of ten CRs.Untreated tumors reached a mean tumor volume of 986 mm³ 19 daysfollowing implant. Tumor growth inhibition of cAC10-E4 was 91% comparedto 90% for cAC10-E8. At 0.25 mg/kg/dose both of these ADCs induced asimilar delay in tumor growth compared to untreated control animals butno complete regressions. cAC10-E2 at 1.0 mg/kg/dose, a dose whichcontained the same amount of MMAE as cAC10-E4 at 0.5 mg/kg/dose andcAC10-E8 at 0.25 mg/kg, induced 10/10 cures. The effect on tumor growthwith cAC10-E2 at 0.5 mg/kg/dose was comparable to that seen withcAC10-E4 and cAC10-E8 at 0.25 mg/kg/dose generating a TGI of 68%. Aphysical mixture of the drug MMAE with cAC10, equivalent to cAC10-E8 atthe 0.5 mg/kg dose, produced only a slight delay in tumor growthcompared to untreated, highlighting that the linkage of drug to antibodyis critical for achieving anti-tumor activity.

Single dose treatment of cAC10-E2, cAC10-E4, and cAC10-E8 in this samemodel were then compared at 1.0 mg/kg (FIG. 12B). Five of six animalstreated with cAC10-E8 achieved cures. Of the six animals treated withcAC10-E4 all achieved complete regressions with five cures out to 108days, the end of the study, one animal was found dead on day 72 with nosign of tumor mass. Even though cAC10-E2 at 1.0 mg/kg contained half asmuch MMAE as cAC10-E4 four of six mice achieved CRs. The control groupconsisting of 1 mg/kg of cAC10 plus 0.037 mg/kg free MMAE, equivalent tothe amount of drug contained in 1 mg/kg of cAC10-E8, had little effecton tumor growth compared to untreated mice.

The initial conjugation of the partially-loaded ADC resulted in amixture of species containing 0-8 drugs/mAb. To evaluate the activity ofthis mixture (cAC10-E4-Mixture) single dose anti-tumor activity ofcAC10-E4-Mixture was compared to the purified cAC10-E4 at 1.0 mg/kg.Similar to the previous single dose experiment nine of ten mice treatedwith cAC10-E4 achieved CRs. Complete regression were generated in eightof ten mice treated with cAC10-E4-Mixture, with an average molar ratioof 4.0. Although it contained a population of ADCs with different drugloadings the partially loaded cAC10-E4-Mixture demonstrated equivalentanti-tumor activity to the purified cAC10-E4.

Maximum tolerated dose and therapeutic window. The single-dosetolerability of cAC10-E2, cAC10-E4, and cAC10-E8 was evaluated in BALB/cmice with three per group. The maximum tolerated dose (MTD) was definedas the highest dose that did not induce greater than 20% weight loss orsevere signs of distress or overt toxicities in any of the animals. ForcAC10-E8, mice were dosed at 10 mg/kg intervals from 30-60 mg/kg. At adose of 50 mg/kg, mice had a maximum weight loss of 14% six days afterinjection, after which the weight loss recovered. A dose of 60 mg/kginduced 23% weight loss six days post injection in one animal. WithcAC10-E4 at 100 mg/kg, mice reached a maximum weight loss ofapproximately 10%. At 120 mg/kg of cAC10-E4 one animal displayed signsof significant distress and 17% weight loss and the animal waseuthanized. Mice treated with cAC10-E2 at doses up to 250 mg/kg, thehighest dose tested, experienced a maximum weight loss of 10.5% 6 dayspost injection, with no signs of distress. Based on our observations,the MTD of cAC10-E2 was at least 250 mg/kg, cAC10-E4 was 100 mg/kg, andcAC10-E8 was 50 mg/kg. Therapeutic index was defined as ratio of thesingle dose MTD to the multi-dose efficacious dose, yielding 100 forcAC10-E8, 200 for cAC10-E4, and at least 250 for cAC10-E2.

Pharmacokinetics. SCID mice were administered with cAC10, cAC10-E2,cAC10-E4, and cAC10-E8 to determine how drug loading effectspharmacokinetics. Table 6 illustrates the pharmacokinetic parametersestablished by non-compartmental analysis.

TABLE 6 Pharmacokinetic parameters of cAC10 and cAC10 ADCs in SCID miceat a dose of 10 mg/kg. t½ AUC CL Vz Name days (μg-day/mL) (mL/day/kg)(mL/kg) cAC10 16.7 2638 3.8 91 cAC10-E2 16.9 2313 4.4 107 cAC10-E4 14.01689 6.0 121 cAC10-E8 14.9 520 19.2 414 The half-life (t½), area underthe curve (AUC), clearance (CL), volume of distribution (Vz), and areaunder the curve from injection to day 14 (AUC t(0-14d)) were calculatedusing non-compartmental analysis.

The time-concentration curves of cAC10, cAC10-E2, cAC10-E4, and cAC10-E8followed bi-exponential declines. The terminal half-lives were 16.7,16.9, 14.0 and 14.7 days, respectively and thus, did not directlycorrelate with drug loading. However, the exposure of ADCs as determinedby the AUC increased as drug loading decreased, ranging from 2638μg-day/mL for unmodified cAC10 to 520 μg-day/mL for cAC10-E8.Conversely, the clearance values increased from 3.8 mL/day/kg for cAC10to 4.4, 6.0 and 19.2 mL/day/kg for cAC10-E2, cAC10-E4, and cAC10-E8,respectively. Similarly, the volume of distribution was found todirectly correlate to drug loading.

Example 9 of Further Study of In Vivo Efficacy

In vivo efficacy experiments were performed using the Karpas 299 CD30positive cell line and are shown in Table 7. Subcutaneous Karpas-299tumors were grown in C.B.-17 SCID mice, with the test articlesadministered when tumors reached approximately 100 mm3 (length×width²).Animals were separated into groups of 5-10 animals, and each group wasinjected with ADC intravenously. Doses were made in 2-fold serialdilutions (0.5, 1.0 2.0 mg/Kg) and tumors that regressed to anunmeasurable size were defined as complete remissions. The dose thatyielded ≧80% complete remissions over several experiments (3-8, with5-15 animals per dose except E6P, which was a single group of 4 animals)was assigned as the efficacious dose. For all the ADCs tested, theefficacious dose was 1 mg/Kg, despite the fact that the amount ofinjected drug component changed with the drug loading. Within theprecision of the experiment (2-fold serial dilutions), the isomericdistribution of the drugs did not influence the efficacy.

To evaluate the tolerability of the ADCs BALB/c mice (parent strain ofthe C.B.-17 SCID) were administered with ADCs. Animal weights weremeasured and clinical observations were recorded over a 14 day period.The MTD was assigned as the highest single dose administered to a BALB/cmouse that did not result in weight loss ≧20% or show signs of distress.For E8, doses were 40, 50 and 60 mg/Kg, for E4, doses were 80, 100 and120 mg/Kg, and for E2, doses were 200 and 250 mg/Kg. As observedpreviously (see Example 8), the absolute drug loading level didinfluence the MTD, with higher drug loading levels having lower MTDvalues. For E4 pure made by DTNB partial reoxidation, the MID wasslightly higher than for E4 pure made by the DTT partial reduction (120versus 100 mg/Kg).

TABLE 7 Mouse in vivo efficacy on CD30⁺ Karpas 299 cells and MTD forcAC10-vcMMAE. ADC Efficacious dose (mg/Kg)^(a) MTD (mg/Kg)^(a) E2P DTT1 >250 E4P DTT 1 100 E4P DTNB 1 120 E6P DTT 1 80 E8 1 50 ^(a)In vivodoses were based on mg of antibody component per Kg of body weight.

No license is expressly or implicitly granted to any patent or patentapplications referred to or incorporated herein. The discussion above isdescriptive, illustrative and exemplary and is not to be taken aslimiting the scope defined by any appended claims.

Various references, including patent applications, patents, andscientific publications, are cited herein, the disclosures of each ofwhich is incorporated herein by reference in its entirety.

1-75. (canceled)
 76. A method for the treatment of cancer, immunedisease, autoimmune disease or infectious disease in a patient,comprising administering to the patient an amount of a modified protein,which is an antibody, comprising: points of conjugation for a cytotoxicor cytostatic agent, wherein at least one point of conjugation for thecytotoxic or cytostatic agent on the antibody can be readily assigned,and wherein less than all possible points of conjugation are availablefor conjugation to the cytotoxic or cytostatic agent.
 77. A method forthe treatment of cancer, immune disease, autoimmune disease orinfectious disease in a patient, comprising administering to the patientan amount of a modified protein, which is a partially loaded, modifiedprotein, comprising: a binding region for interaction with a bindingpartner; at least two points of conjugation having a similaraccessibility or activability for conjugation of a drug or label bychemical means; at least two drugs or labels, each drug or labelcovalently linked to one point of conjugation; wherein less than all ofthe possible points of conjugation having a similar accessibility oractivability are linked to a drug or label.
 78. The method of claim 77,wherein: the modified protein is an antibody; the binding region is anantigen-binding domain of the antibody; the points of conjugation arethiol groups; and the antibody comprises at least one interchaindisulfide bond.